Application of pleuromutilin antibiotics in the preparation of drugs for anti-tumor and reversal of drug resistance to molecular targeted therapy

Abstract

The invention belongs to the field of medicine, and relates to the application of pleuromutilin antibiotics in the preparation of anti-tumor and molecular targeted therapy resistance reversal drugs; in HepG2 liver cancer cells, Lefamulinacetate can promote the liver cancer cell apoptosis-promoting and proliferation-inhibiting effects caused by sorafenib, and increase the cell killing effect of sorafenib; in a nude mouse subcutaneous transplanted tumor model, Lefamulinacetate can increase the efficacy of inhibiting tumor growth in vivo caused by sorafenib, reduce tumor weight and the percentage of cell death area in tumor tissue and the number of KI67 positive cells, but has no effect on the nude mouse body weight, plasma ALT, AST, BUN, CRE content, and no damage to the nude mouse heart, liver, spleen, lung, kidney and other organs, indicating that Lefamulinacetate has a good effect of reversing the sorafenib resistance of hepatocellular carcinoma; Lefamulinacetate has the advantages of good effect and high safety, and gives full play to the advantages of new uses of old drugs, and is expected to be developed into an effective drug for treating molecular targeted therapy resistant hepatocellular carcinoma.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the application of pleuromutilin antibiotics in the preparation of drugs for anti-tumor and reversal of molecular targeted therapy resistance. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary liver tumor, accounting for 70% to 90% of primary liver cancers, and the mortality rate ranks second. The global incidence continues to rise, seriously threatening people's lives and health. At present, surgical treatment represented by liver resection, liver transplantation and local ablation is the first choice and the most effective measure for treating early HCC patients. However, due to the difficulty of early diagnosis, rapid disease progression, high recurrence and metastasis rate and lack of effective treatment methods, most HCC patients are diagnosed in the middle and late stages. Patients with middle and late stage liver cancer and most liver cancer patients with poor liver function are not suitable for surgical resection. They can only prolong their survival time through transcatheter arterial chemoembolization (TACE), radiotherapy and chemotherapy.

[0003] Sorafenib is a multi-target tyrosine kinase inhibitor and the first molecular targeted drug approved by the FDA for the treatment of advanced HCC. It can directly inhibit the proliferation of tumor cells by blocking the cell signaling pathway mediated by RAF / MEK / ERK, and can also indirectly inhibit the growth of tumor cells by blocking the formation of tumor angiogenesis by inhibiting VEGFR and platelet-derived growth factor (PDGF) receptors. It has shown good anti-HCC effects in both in vivo and in vitro studies and promotes the survival of patients with advanced liver cancer. However, the emergence of primary and acquired drug resistance in clinical treatment not only limits the long-term benefits of HCC patients from sorafenib treatment, but also worsens the disease and accelerates the course of the disease. Primary and acquired resistance to sorafenib in HCC involves multiple mechanisms, including autophagy, epithelial-mesenchymal transition, cancer stem cells, tumor microenvironment and epigenetic regulation, and may involve many signaling pathways, such as Wnt / β-catenin, TGFβ, Ras / MEK / ERK, PI3K / Akt, TNFα / NF-κB and JAK / STAT pathways. However, the mechanism of sorafenib resistance has been less studied in vivo and in HCC patients. Therefore, it is of great clinical significance to explore the problem of sorafenib resistance in HCC and seek possible reversal strategies.

[0004] The development cycle of new drugs is long, the investment is large, and the risk is high. "New uses of old drugs" can make up for these limitations to a certain extent. For drug discovery for specific diseases, taking chemical small molecule drugs as an example, from obtaining active lead compounds in in vitro cell models to finally moving towards clinical application, it is necessary to survive the difficult process of "nine deaths and one life" such as multi-level activity and efficacy evaluation, targeting and mechanism of action exploration, toxicity and safety evaluation, and metabolic performance evaluation. However, old drugs that have survived many levels of tests can at least quickly meet the basic conditions for drug safety evaluation and metabolism, so they are expected to accelerate the process of new drug discovery. Optimization based on old drugs as the skeleton can greatly shorten the preclinical research cycle of lead compound discovery, lead compound optimization, and even candidate drugs. At the same time, based on clear pharmacokinetic properties and safety parameters, the risk of clinical research failure can be effectively reduced. At present, many achievements have been made in the research on "new uses of old drugs", such as aspirin's new uses in anti-cancer, prevention of Alzheimer's disease, lowering blood sugar, etc.; however, the current research on drugs to reverse sorafenib-resistant hepatocellular carcinoma is mostly limited to natural products, and there are few clinical studies on reversing hepatocellular carcinoma resistance, and the mechanism of action is still unclear.

[0005] Lefamulin acetate is a pleuromutilin antibiotic and an FDA-approved drug for the treatment of community-acquired bacterial pneumonia (CABP). Its mechanism of action is that Lefamulin acetate inhibits protein synthesis by binding to the peptidyl transferase center of the 50S bacterial ribosome, thereby preventing the binding of transfer RNA to peptide transfer and achieving antibacterial effects.

[0006]

[0007] Based on the constructed liver cancer sorafenib-resistant cell line HepG2SR, the present invention conducts a screening study on the sorafenib sensitization activity of drugs in the FDA-approved clinical drug library that have been marketed, and finds that the pleuromutilin antibiotic drugs Valnemulin HCl, Retapamulin, and Lefamulin acetate all have different degrees of sensitization effects, indicating that this type of parent core drug has the potential to reverse the sorafenib resistance of hepatocellular carcinoma. In view of the limitations of the former two as veterinary drugs and topical drugs, this laboratory takes Lefamulin acetate as the research object, conducts an in-depth study on its reversal of sorafenib resistance in hepatocellular carcinoma, and further verifies the effect of Lefamulin acetate on increasing the in vivo sorafenib efficacy in the HepG2 subcutaneous heterotopic transplant tumor model from an in vivo animal model on the basis of investigating the safety of the drug. The drug Lefamulin acetate currently has fewer adverse reactions in the clinical treatment process, and its effect of reversing the molecular targeted therapy resistance of hepatocellular carcinoma has not been reported in relevant literature at home and abroad so far. Summary of the invention

[0008] The present invention uses tumor cell models and mouse tumor models to discover a new use of pleuromutilin antibiotic Lefamulinacetate in reversing drug resistance of molecular targeted therapy of hepatocellular carcinoma by directly binding to ILF3 protein.

[0009] Application of pleuromutilin antibiotics in the preparation of drugs for reversing drug resistance in molecular targeted therapy of liver cancer.

[0010] The liver cancer is a liver cancer that is insensitive or resistant to sorafenib treatment.

[0011] The pleuromutilin antibiotic is Valnemulin HCl, Retapamulin or Lefamulin acetate.

[0012] A pharmaceutical composition is used in a drug for treating and reversing drug resistance of liver cancer treated with sorafenib, characterized in that the pharmaceutical composition comprises lefamulin acetate or a pharmaceutically acceptable salt or ester thereof and sorafenib, a clinical molecular targeted therapeutic drug for hepatocellular carcinoma.

[0013] When the present invention is used in combination with sorafenib to treat hepatocellular carcinoma, the clinically available drug Lefamulinacetate is safe and effective for oral or parenteral administration. The oral drug is a tablet; the parenteral drug is an injection.

[0014] The dosage of the pleuromutilin antibiotic Lefamulin acetate of the present invention can be adjusted based on comprehensive consideration of factors such as the mode of administration, severity of illness, age of the patient, and the presence or absence of previous medical history.

[0015] Beneficial effects.

[0016] The present invention relates to a new use of Lefamulin acetate. Specifically, the present invention uses a liver cancer sorafenib-resistant cell line HepG2SR as a drug screening cell model, and combines drugs in a clinical drug library approved by the FDA and sorafenib for drug screening. CCK8 is used to determine the cell survival rate, and it is first found that pleuromutilin antibiotics Valnemulin HCl, Retapamulin, and Lefamulin acetate all have different degrees of sensitization effects on sorafenib. In view of the convenience and practicality of the administration method and the patient's medication, Lefamulin acetate is selected as a further research object. The present invention uses HepG2 cells to conduct in vitro activity studies. In the cells, the cells are divided into a control group, a sorafenib administration group, a Lefamulin acetate administration group, and a sorafenib and Lefamulin acetate combined administration group. After 48 hours of administration, the HepG2 cells are subjected to Annexin-V / PI double-staining flow cytometry to detect the cell apoptosis rate; CCK8, EDU, and clone formation are used to detect cell proliferation.

[0017] In order to further evaluate the drug's ability to increase the anti-liver cancer effect of sorafenib in vivo, the present invention uses HepG2 cells to construct a nude mouse subcutaneous transplant tumor model to confirm the pharmacological activity of Lefamulin acetate in increasing the effect of sorafenib in treating hepatocellular carcinoma. In the animal model, 4-6 week-old Balb / c nude mice were used, and HepG2 cells were inoculated in the flanks. When the tumor was palpable, the drug was administered and divided into a solvent group, a sorafenib administration group, a Lefamulin acetate administration group, and a sorafenib and Lefamulin acetate combined administration group. After 3 weeks of administration, the blood of the nude mice was collected for biochemical index analysis; the heart, liver, spleen, lung, and kidney of the nude mice were collected for HE staining; the tumor tissues of the nude mice were collected for HE staining and KI67 immunohistochemical staining; and the ALT, AST, BUN, and CRE levels in the blood of the nude mice were detected.

[0018] To further explore the mechanism by which lefamulin acetate enhances the sensitivity of sorafenib, protein spectrum analysis was used to identify the proteins bound by lefamulin acetate, and Docking, DARTS, and CETSA were used to confirm the binding of lefamulin acetate to ILF3. Western-Blot was used to detect the effect of lefamulin acetate on ILF3 protein.

[0019] Results from cell and animal models show that:

[0020] 1. The apoptosis rate in cells co-administered with Lefamulin acetate and sorafenib was significantly increased compared with that in cells administered with sorafenib alone, indicating that Lefamulin acetate can enhance the effect of sorafenib in promoting apoptosis of liver cancer cells.

[0021] 2. The proliferation rate of cells co-administered with Lefamulin acetate and sorafenib was slower, and the number of EDU-positive cells and clone formation was significantly reduced compared with sorafenib alone, indicating that Lefamulin acetate can enhance the inhibitory effect of sorafenib on liver cancer cell proliferation.

[0022] 3. In the animal model, there was no significant weight loss in each group of mice, and no obvious damage to the heart, liver, spleen, lung, and kidney organs occurred. There was no significant difference in the levels of plasma ALT, AST, BUN, and CRE. There were no signs of poisoning and no death occurred, indicating that the drug is relatively safe in the tumor model. This not only provides a basis for the in vivo pharmacodynamic evaluation of the compound, but also lays the foundation for the development of new clinical uses of this type of drug.

[0023] 4. In vivo pharmacodynamics showed that the tumors of nude mice co-administered with Lefamulin acetate and sorafenib grew more slowly and weighed less. The results of tumor tissue pathological section staining showed that Lefamulin acetate and sorafenib synergistically increased the percentage of liver cancer cell death area and reduced the number of KI67-positive cells, indicating that the proliferation of liver cancer cells in vivo was inhibited, indicating that Lefamulin acetate can treat HCC by increasing the sensitivity of sorafenib.

[0024] 5. Mechanism exploration showed that Lefamulin acetate directly binds to the ILF3 protein, but has no effect on the expression level of ILF3. The drug can reduce the expression of ILF3 downstream target genes, suggesting that Lefamulin acetate may directly bind to the ILF3 target protein to block its binding to the promoter region of the downstream drug-resistant target gene, thereby inhibiting its transcriptional activity and exerting a sensitizing effect.

[0025] Therefore, the present invention provides pleuromutilin antibiotic Lefamulin acetate, which has good market prospects and clinical value in terms of safety and effectiveness in reversing drug resistance in molecular targeted therapy of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 IC50 results after administration of different concentrations of sorafenib in HepG2, HepG2SR (sorafenib-resistant) cells.

[0027] Figure 2 , IC50 results of HepG2SR cells after administration of different concentrations of sorafenib in the presence or absence of Lefamulin acetate. A is Valnemulin HCl, B is Retapamulin, C is Lefamulin acetate (10μM), D is Lefamulin (10 and 20μM), and E is Retapamulin (10 and 20μM).

[0028] Figure 3 The cell apoptosis rates in HepG2 cells were measured after 48 hours of treatment with DMSO, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate, respectively.

[0029] Figure 4 Cell absorbance values ​​in HepG2 cells 24, 48, and 72 hours after administration of DMSO, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate, respectively.

[0030] Figure 5 The results of EDU staining of HepG2 cells 24 hours after administration of DMSO, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate.

[0031] Figure 6The results of cell clone formation were analyzed by administering DMSO, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate to HepG2 cells for 24 hours and then culturing for 2 to 3 weeks.

[0032] Figure 7 Body weight change curve of nude mice with subcutaneous tumor transplantation after being given Vehicle, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate for 3 weeks.

[0033] Figure 8 HE staining images of heart, liver, spleen, lung and kidney tissues of nude mice with subcutaneous tumor transplantation were given Vehicle, sorafenib, Lefamulin acetate and sorafenib + Lefamulin acetate respectively for 3 weeks.

[0034] Fig. 9 , Plasma ALT (A), AST (B), CRE (C), and BUN (D) levels in nude mice with subcutaneous tumor transplantation after being given Vehicle, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate for 3 weeks, respectively.

[0035] Fig.10 Tumor growth curve (A), tumor volume change (B), and tumor weight (C) of nude mice with subcutaneous tumors given Vehicle, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate, respectively, for 3 weeks.

[0036] Fig.11 HE staining images of tumor tissue sections (A) and KI67 immunohistochemical staining images (B) of nude mice with subcutaneous tumors were given Vehicle, sorafenib, Lefamulin acetate, and sorafenib + Lefamulin acetate, respectively, for 3 weeks.

[0037] Fig.12 , direct binding indications of Lefamulin acetate and ILF3 protein, including Docking (A), DARTS (B), and CETSA (C).

[0038] Fig.13, Effect of Lefamulin acetate on ILF3 protein expression (A), Effect of Lefamulin acetate on the mRNA levels of ILF3 downstream target genes (B is IL-2, C is IL13, and D is Sunvivin. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and examples, but the contents described are intended to explain the present invention rather than to limit it. Unless otherwise specified, the raw materials and reagents involved in the experimental methods in the following examples are all common commercial products and can be purchased on the market.

[0040] Example 1 Screening of drugs for reversing drug resistance in hepatocellular carcinoma

[0041] 1. Experimental Methods

[0042] The present invention constructs a sorafenib-resistant liver cancer cell line by using a drug concentration gradient increase method and a continuous drug administration method. The specific process is to first stimulate the cells with sorafenib at an IC50 concentration, and then replace the medium containing the drug every day to continue culturing. After about 2 to 4 weeks of normal cell growth, the sorafenib concentration is increased by a gradient of 0.5 μM. After about 3 to 6 months, the drug-resistant strain is successfully constructed, and the successful construction of the drug-resistant strain is characterized by the CCK8 method. After the drug-resistant strain is successfully constructed, it is cultured at 37°C and 5% CO2. After the cell confluence is about 90%, about 1×10 4 The cells were plated in a 96-well plate, and the culture medium was aspirated after the cells adhered to the wall. The culture medium containing the drug was added, and the cells were grouped into a group using the FDA-approved clinical drug library in combination with sorafenib and a group using sorafenib alone. CCK8 was added 48 hours after administration, and the cells were incubated at 37°C in the dark for 2 to 4 hours, and the absorbance at 450nm was measured by an enzyme reader to monitor cell viability. By comparing the combination drug group with the sorafenib alone group, the drug that sensitizes to sorafenib was found.

[0043] 2. Experimental results

[0044] The present invention first successfully constructed a liver cancer resistant cell line ( Figure 1), the drug resistance index RI was 2.78; and it was found that the pleuromutilin antibiotics Valnemulin HCl, Retapamulin, and Lefamulin acetate could significantly reduce the survival rate of liver cancer cells in the drug-resistant strains. Considering the convenience and practicality of clinical medication, Lefamulin acetate was selected for further study. By further measuring the IC50 value, it was found that the sorafenib IC50 value was significantly reduced after combined use, and the drug combination index (CI) was less than 1, indicating that Lefamulin acetate can increase the sensitivity of sorafenib to kill liver cancer cells ( Figure 2 ), and further studies have found that Lefamulin acetate also has a sensitizing effect on two other molecular targeted therapeutic drugs for liver cancer, Levatinib and Regorafenib ( Figure 2 ).

[0045] Example 2 Study on the in vitro sensitization activity of Lefamulin acetate on liver cancer cells

[0046] 1. Experimental Methods

[0047] 1.1 Annexin V-PI cell apoptosis rate determination

[0048] The cell apoptosis rate was determined using the Annexin V-PI cell apoptosis kit. HepG2 cells were plated at approximately 3 × 10 5 Cells were seeded into 6-well plates and treated with lefamulin acetate and sorafenib for 48 hours after the cells adhered overnight. After the cells were collected, they were incubated with FITC-conjugated Annexin V and PI at room temperature in the dark for 15 minutes, and the cell apoptosis rate was analyzed by flow cytometry.

[0049] 1.2 Cell proliferation curve determination

[0050] HepG2 cells were grown at approximately 5 × 10 3 Cells were inoculated into 96-well plates, and drugs were added after the cells adhered overnight. The absorbance values ​​were measured using CCK8 at 24h, 48h, and 72h.

[0051] 1.3 EDU infiltration experiment

[0052] HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged, and diluted with complete medium. The cells were counted using a cell counter and the cell density was adjusted to about 1.0 × 10 5cells / mL, and then 100μL was inoculated into a 96-well plate per well. After culturing in a 37°C, 5% CO2 incubator for 24h, EdU was diluted with serum-free medium into a 20μM solution and added to the culture plate for incubation for 6h. The culture medium was discarded, and the cells were carefully washed 3 times with PBS. After fixing with 4% paraformaldehyde in a fume hood for 15min, the fixative was discarded, washed, and incubated with permeabilization solution (0.3% Triton X-100 in PBS) at room temperature for 10min. The permeabilization solution was removed, and the cells were washed 1-2 times with 0.1ml washing solution per well for 3-5 minutes each time, and then 50μL Click reaction solution was added to each well, and the culture plate was gently shaken to ensure that the reaction mixture could evenly cover the sample. Incubate at room temperature in the dark for 30min, remove the Click reaction solution, wash 3 times with washing solution, incubate with Hochest33342 staining solution at room temperature for 10min, wash 3 times with washing solution, and then take photos for detection using high content.

[0053] 1.4 Clone formation assay

[0054] HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin, the digestion solution was discarded, and the cells were gently pipetted and mixed with complete culture medium. The cells were counted and the cell density was adjusted to 2×10 3 cells / mL, and then 2mL per well was inoculated in a 6-well culture plate, incubated in an incubator, the culture medium was renewed every 2-3 days, and the clone formation process was observed under an inverted microscope. About two weeks later, the culture was terminated when clones visible to the naked eye appeared in the culture plate, the culture medium was discarded, and after washing with PBS twice, 4% paraformaldehyde was fixed for 15 minutes, the fixative was discarded, and PBS was used to wash twice, 1mL of crystal violet staining solution was added to each well for staining for about 20 minutes, and finally PBS was used to wash away the excess staining solution, the well plate was placed in the air to dry, and photos were taken.

[0055] 2. Experimental results

[0056] The results showed that the apoptosis rate of cells in the group treated with lefamulin acetate and sorafenib was significantly increased ( Figure 3 ), the proliferation rate slowed down ( Figure 4 ), the number of EDU-positive cells decreased ( Figure 5 ), the number of clones formed increased ( Figure 6 ), indicating that Lefamulinacetate can significantly increase the apoptosis-promoting and anti-proliferation effects of sorafenib on liver cancer cells, and has a certain sensitizing activity.

[0057] Example 3 Lefamulin acetate increases the anti-HCC efficacy of sorafenib in vivo

[0058] 1. Experimental Methods

[0059] 1.1 Method for constructing nude mouse subcutaneous transplant tumor model

[0060] A sufficient amount of HepG2 cells were cultured in a cell culture incubator. The cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were counted, and the cell pellet was washed twice with PBS. Then, the cells were resuspended in a mixture of serum-free culture medium and Matrigel matrix gel (1:1) to a cell density of 2×10 7 cells / mL of cell suspension, mix thoroughly and place on ice. The nude mouse tumor model was inoculated subcutaneously. The right posterior groin of athymic male BALB / c nude mice was used as the tumor puncture site. The puncture site was disinfected with 70% alcohol, the skin of the nude mouse was lifted, and 0.2mL of cell suspension was quickly injected into the groin site with a 1mL sterile microsyringe.

[0061] 1.2 Observation of tumor formation and tumor growth

[0062] After cell inoculation, the tumor site grows to about 5mm 3 The tumor was considered to be a tumor, which means the model was successfully established. The longest diameter (L) and the shortest diameter (W) perpendicular to the subcutaneous transplanted tumor were measured every 2 to 3 days with a digital caliper, and the average tumor volume (Tumor Volume, TV) was calculated: TV = 0.5 × L × W 2 The tumor volume is about 100mm 3 The random grouping and drug administration began at 14:00 pm, with 6 mice in each group, divided into the solvent group (oral administration of castor oil: 95% ethanol: water = 1:1:6, intraperitoneal injection of normal saline), the sorafenib group (30 mg / kg / day), the low-dose intraperitoneal injection of Lefamulin acetate group (25 mg / kg / day), the high-dose intraperitoneal injection of Lefamulin acetate group (50 mg / kg / day), the sorafenib group (30 mg / kg / day) + the low-dose intraperitoneal injection of Lefamulin acetate group (25 mg / kg / day), and the sorafenib group (30 mg / kg / day) + the high-dose intraperitoneal injection of Lefamulin acetate group (50 mg / kg / day). The growth curve of transplanted tumors was drawn according to the tumor volume. The experiment was ended 3 weeks after administration. The mice were fasted but not watered for 12 h before sampling and anesthetized with isoflurane inhalation. Blood was collected from the eyeballs and the mice were killed by cervical dislocation. Tumor tissues were removed, weighed, and photographed. Part of the tissue was fixed by immersion in 4% paraformaldehyde solution and embedded in paraffin for H&E staining and immunohistochemical staining of pathological sections. The other part was quickly frozen in liquid nitrogen and placed in a -80°C refrigerator for subsequent experimental detection.

[0063] 1.3 Plasma sample collection

[0064] After the mice were anesthetized, their eyes were removed and blood was collected in 1.5 ml EP tubes (with sodium heparin anticoagulant). After standing for 30 min, the blood was centrifuged at 4000 rpm for 10 min at 4°C. The upper transparent plasma was gently transferred and stored at -80°C for subsequent biochemical index detection.

[0065] 2. Experimental results

[0066] 2.1 Lefamulin acetate is non-toxic to nude mice

[0067] After the administration began, the weight of the nude mice, their physical condition (such as whether they were short of breath, whether they had bleeding spots on their skin, etc.), and whether they died were recorded every day. After the administration, the plasma and organs of the nude mice in each group were collected, and the plasma biochemical indicators were tested. The pathological staining of the organs was observed. The results showed that there was no significant decrease in the weight of the mice in each group after the administration ( Figure 7 ), plasma biochemical indicators showed that the liver function (ALT, AST) and renal function (CRE, BUN) of nude mice in each group were normal ( Figure 8 ), HE staining results showed no signs of poisoning in various organs and no death occurred ( Fig. 9 ), indicating that the drug is relatively safe.

[0068] 2.2 Lefamulin acetate can significantly increase the anti-liver cancer effect of sorafenib in vivo

[0069] The tumor growth curve and tumor weight results showed that the tumor growth of nude mice in the group treated with Lefamulin acetate and sorafenib was slower ( Fig.10 ), the tumor weight is lighter ( Fig.11 ), the results showed that the combined administration of Lefamulin acetate and sorafenib could synergistically inhibit the growth of liver cancer in vivo. The results of HE staining of tumor tissue and KI67 immunohistochemical staining showed that the combination of Lefamulin acetate and sorafenib could synergistically increase the percentage of liver cancer cell death area and inhibit the proliferation of liver cancer cells in vivo (the number of KI67 positive cells decreased), indicating that Lefamulin acetate can increase the efficacy of sorafenib.

[0070] Example 4 Target protein study of Lefamulin acetate

[0071] 1. Experimental Methods

[0072] 1.1 Molecular Docking Simulation

[0073] Molecular docking simulations were performed using the Schrödinger model using a docking algorithm based on the ILF3 protein structure (PDB: 2L33) and the structural formula of Lefamulin acetate.

[0074] 1.2 Drug Affinity Response Target Stability (DARTS)

[0075] Cells were harvested and total protein was isolated using lysis buffer. Cell lysates were centrifuged at 18,000 × g for 10 min at 4 °C. The supernatant was diluted 1:10 with 10 × TNC buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM CaCl 2) and treated with different concentrations of Lefamulin acetate or DMSO as controls. After incubation at room temperature for 2 h, pronase was added and incubated for another 5 min at 37 °C. The reaction was stopped by adding protease inhibitors and SDS-PAGE loading buffer followed by immunoblotting.

[0076] 1.3 Cellular thermal shift assay (CETSA)

[0077] HepG2 cells were cultured in 10 cm dishes until 70-80% confluence. The cells were then treated with Lefamulin acetate or DMSO and incubated for 6 h. The cells were collected, washed with PBS, and resuspended to 5 × 10 cells / mL in PBS supplemented with protease inhibitors. 6 Cells / ml. Take 100 μl of each cell suspension and dispense it into PCR tubes, heat it at 40-68°C for 3 minutes in a thermal cycler, and immediately freeze-thaw and lyse in liquid nitrogen. The cell lysate was clarified by centrifugation at 15,000×g for 15 minutes at 4°C. The supernatant was analyzed by immunoblotting.

[0078] 1.4 ILF3 protein expression assay

[0079] HepG2 cells were inoculated into a cell culture dish, and drugs were added after the cells adhered overnight. The cells were collected after 48 hours, and the expression of ILF3 in the cells was detected by Western Blot according to the instructions of the protein extraction kit.

[0080] 2. Experimental results

[0081] Molecular docking simulation, DARTS, and CETSA results showed that Lefamulin acetate directly bound to ILF3 protein with a score of -7. Fig.12 ), Western Blot results showed that Lefamulin acetate had no effect on the protein expression of ILF3 ( Fig.13), indicating that Lefamulin acetate sensitizes sorafenib by directly binding to ILF3. The specific mechanism is that after the drug binds to ILF3, it blocks the binding of ILF3 to the promoter region of its downstream drug-resistant target gene, thereby inhibiting its transcriptional activity and exerting a sensitizing effect.

Claims

1. The use of pleuromutilin antibiotics in the preparation of anti-tumor drugs, characterized in that: The tumor is liver cancer; the pleuromutilin antibiotic is Lefamulin acetate 2. The use of pleuromutilin antibiotics in combination with molecular targeted anti-tumor drugs in the preparation of anti-tumor drugs, characterized in that: The tumor is liver cancer; the molecular targeted anti-tumor drug is sorafenib, levatinib, or regorafenib; the pleuromutilin antibiotic is Valnemulin HCl, Retapamulin, or Lefamulin acetate 3. Use of pleuromutilin antibiotics in the preparation of drugs for reversing molecular targeted anti-tumor drug resistance, characterized in that: The tumor is liver cancer; the molecular targeted anti-tumor drug is sorafenib, levatinib, or regorafenib; the pleuromutilin antibiotic is Valnemulin HCl, Retapamulin, or Lefamulin acetate

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