Use of oligomycin A in the preparation of drugs for competitively inhibiting EphB3 to reverse loratinib resistance

Abstract

This invention discloses the use of oligomycin A in the preparation of a drug for competitively inhibiting EphB3 to reverse lorlatinib resistance, belonging to the field of biomedical technology. This invention experimentally discovered that EphB3 is a key factor mediating lorlatinib resistance in lung cancer, and that existing oligomycin A can effectively reverse EphB3-mediated resistance by competitively binding to EphB3. Based on this, this invention protects the use of oligomycin A in the preparation of a drug for reversing lorlatinib resistance in tumors (especially ALK-positive non-small cell lung cancer). Simultaneously, this invention also protects a therapeutically effective pharmaceutical composition comprising oligomycin A and lorlatinib, and a non-therapeutic method for reversing cellular resistance to lorlatinib in vitro. This invention provides a novel and effective solution for overcoming lorlatinib resistance in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the use of oligomycin A in the preparation of a drug for competitively inhibiting EphB3 to reverse loratinib resistance. Background Technology

[0002] ALK gene rearrangement is a key driver gene in non-small cell lung cancer (NSCLC), accounting for approximately 3% to 5% of lung adenocarcinomas. This mutation drives tumor development and progression by continuously activating downstream signaling pathways, and is often referred to as a "diamond mutation" due to its good response to targeted therapy. However, patients carrying this mutation often have adverse clinical characteristics such as high malignancy and a high risk of distant metastasis.

[0003] ALK tyrosine kinase inhibitors (ALK-TKIs), represented by lorlatinib, have brought significant survival benefits to these patients and have become an important cornerstone of clinical treatment. However, unlike traditional chemotherapy, targeted therapy cannot avoid the development of drug resistance, which has become a major bottleneck restricting its long-term efficacy. Clinically, after patients develop resistance to first- and second-generation ALK-TKIs, they often continue treatment by sequentially using next-generation drugs (such as lorlatinib). However, tumor cells can also develop adaptive resistance to lorlatinib, leading to treatment failure. More challenging is that tumors that have undergone multiple lines of ALK-TKI therapy often have more complex and diverse resistance mechanisms, and often lack effective subsequent targeted therapy strategies, leaving patients facing a dilemma of having no available drugs.

[0004] Currently, the resistance mechanism to loratinib is not fully understood, and exploring its resistance pathways and finding effective reversal strategies are major challenges that urgently need to be addressed in clinical practice. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the primary technical problem solved by this application is to provide the use of oligomycin A in the preparation of a drug for competitively inhibiting EphB3 to reverse loratinib resistance. A secondary technical problem solved by this application is to provide a pharmaceutical composition for treating ALK-positive non-small cell lung cancer resistant to loratinib.

[0006] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0007] Use of oligomycin A in the preparation of drugs for reversing resistance of tumor cells to loratinib.

[0008] Furthermore, the drug resistance is mediated by EphB3.

[0009] Furthermore, the oligomycin A reverses the drug resistance by competitively binding to EphB3.

[0010] In some embodiments, the tumor cells are ALK-positive cancer cells.

[0011] In some embodiments, the ALK-positive cancer is non-small cell lung cancer.

[0012] A pharmaceutical composition for treating ALK-positive non-small cell lung cancer resistant to loratinib, comprising a therapeutically effective amount of oligomycin A and a therapeutically effective amount of loratinib.

[0013] In some embodiments, the pharmaceutical composition is prepared as a dosage form for simultaneous, separate, or sequential administration.

[0014] In some embodiments, the oligomycin A and loratinib are prepared as a single unit dosage form or as separate dosage forms.

[0015] A method for non-therapeutic reversal of cell resistance to loratinib in vitro, comprising contacting the cells with an effective amount of oligomycin A.

[0016] In some embodiments, the cells are derived from an individual with ALK-positive non-small cell lung cancer that is resistant to loratinib.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) Currently, the main mechanisms of lolatinib resistance include ALK-dependent and ALK-independent pathways. This invention comprehensively utilizes cutting-edge technologies such as protein thermostability experiments, molecular docking, and proteomics to reveal for the first time that EphB3 bypass activation mediates lolatinib resistance, providing new evidence for the mechanism of lolatinib resistance. Furthermore, experiments have confirmed that existing oligomycin A can directly bind to EphB3, inhibiting its downstream signaling pathways and thus reversing the resistance phenotype. This discovery fills a gap in the research on EphB3 in ALK-TKI resistance mechanisms and represents original innovation. This invention provides a solid theoretical basis for subsequent drug development and clinical application.

[0019] (2) The pharmaceutical composition provided by this invention can significantly enhance the inhibitory effect on drug-resistant tumors in cell and animal models, effectively delay disease progression, and demonstrates the clinical application potential of prolonging progression-free survival (PFS) and overall survival (OS). The pharmaceutical composition provided by this invention can be prepared into various dosage forms to meet the clinical medication needs of different patients and has wide applicability.

[0020] (3) This invention provides a novel treatment strategy to overcome loratinib resistance, and offers a feasible solution to the dilemma of sequential treatment failure in ALK-positive NSCLC patients. It is of great significance to promote the development of precision oncology. Attached Figure Description

[0021] Figure 1 Figure A shows the construction and validation of a lorlatinib-resistant cell model. A: MTT assay to detect the effect of lorlatinib on parental cells and resistant cells. Value; BC: Graph showing the effect of loratinib on the clonogenic ability of parental and resistant cells as verified by cell colony formation assay; Note: P < 0.001 indicates a statistically significant difference; NS (No significance) indicates no statistical significance.

[0022] Figure 2 Figure 1 shows the synergistic inhibition of lorlatinib-resistant cell proliferation by oligomycin A and lorlatinib; A: CCK8 assay shows the synergistic effect; B: CCK8, EdU, and cell colony formation assays show the effect of combination therapy on proliferation; Note: P < 0.001, the difference is statistically significant;

[0023] Figure 3 The diagram shows the analytical process for the direct target of oligomycin A; A: Mass spectrometry sample quality control based on CBB gel image; B: TPP analysis of oligomycin A binding proteins; C: Treatment of H3122-LR cells with oligomycin A, followed by transcriptome sequencing and GO and KEGG pathway enrichment analysis; D: The thermostability of EphB3 protein was significantly improved after oligomycin A treatment.

[0024] Figure 4 Structural stability and kinetics of protein-drug complexes; A: RMSD of protein-drug complexes during simulation; B: RMSF spectrum of protein; C: Radius of gyration (Rg) plot; D: Solvent accessible surface area (SASA) of protein-drug interaction interface; E: Number of hydrogen bonds formed between protein and drug; F: Binding free energy spectrum of protein-drug complexes.

[0025] Figure 5 Experimental diagrams illustrating EphB3-mediated ALK-TKI resistance; A: CCK8 assay shows that interfering with EphB3 in H3122-LR cells can reverse lorlatinib resistance; B: After interfering with EphB3, the resistance-reversing effect of oligomycin A is reduced; C: Western blot assay detects the overexpression efficiency of EphB3; D: CCK8 assay shows that overexpression of EphB3 reduces lorlatinib resistance. E: Colony formation assay showed that overexpression of EphB3 enhanced the cell colony formation ability; F: EdU assay showed that overexpression of EphB3 weakened the inhibitory effect of loratinib on cell proliferation, scale bar is 100 μm;

[0026] Figure 6Experimental diagram showing the effect of oligomycin A in reversing loratinib resistance in a nude mouse subcutaneous xenograft tumor model; A: Display of experimental animals in each group; BC: Tumor size and weight in each group; D: Changes in tumor size in each group; E: Changes in body weight of nude mice in each group; Note: P<0.05, P<0.01, P<0.001, the difference is statistically significant. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0028] Example 1

[0029] I. Construction of drug-resistant cell models and reversal of loratinib resistance by oligomycin A

[0030] 1. Materials and Reagents

[0031] (1) H3122 cell line;

[0032] (2) Loratinib;

[0033] (3) Oligomycin A;

[0034] (4) MTT and CCK8 reagent kits, EdU cell proliferation kit, and crystal violet staining solution;

[0035] (5) Culture medium and other conventional cell culture reagents.

[0036] 2. Methods and Steps

[0037] Construction of drug-resistant cell model: H3122 cells were exposed to gradually increasing concentrations of lorlatinib using a concentration gradient escalation method and cultured continuously for several weeks to months until the H3122-LR cell line exhibiting significant resistance to lorlatinib was obtained. The MTT assay was used to determine the drug resistance of H3122-LR cells. The resistance value was calculated, and the resistance multiple was determined. The resistance characteristics were further verified through clonogenic experiments.

[0038] ① Cell viability assay: After 48 h of treatment, cell viability in each group was detected using CCK8 reagent to determine cell survival rate and calculate cell viability. Values. By comparing the combination therapy group with the single-drug group. The changes were investigated to determine the reversal effect of oligomycin A on loratinib resistance and the optimal synergistic concentration.

[0039] ② Cell proliferation capacity assessment: The effects of the drug on cell proliferation capacity were further observed through CCK8 assay, colony formation assay, and EdU assay.

[0040] 3. Results

[0041] Experimental results are as follows Figure 1 and Figure 2 As shown, Figure 1 This indicates that H3122-LR cells respond to loratinib. The levels were significantly higher than those of wild-type H3122 cells, indicating that a drug resistance model was successfully established. Figure 2 This indicates that oligomycin A and loratinib have a synergistic inhibitory effect on proliferation in H3122-LR cells, thereby reversing loratinib resistance.

[0042] II. Identification of the direct target of oligomycin A

[0043] 1. Materials and reagents:

[0044] (1) H3122-LR cells;

[0045] (2) Oligomycin A;

[0046] (3) siRNA-EphB3;

[0047] (4) Related antibodies and Western Blot reagents.

[0048] 2. Method and steps:

[0049] (1) Protein thermostability analysis (TPP): H3122-LR cell lysates were treated at different temperature gradients (37℃ to 67℃), and soluble protein components were separated by centrifugation. The content of soluble proteins at each temperature point was quantitatively detected by Western blotting and mass spectrometry (Label-free + DIA), and protein thermostability curves were plotted. Proteins with significantly improved thermostability after binding to oligomycin A were screened, with a focus on screening candidate targets with ΔTm > 5℃.

[0050] (2) Transcriptome sequencing: H3122-LR cells were treated with 10 μmol / L oligomycin A for 48 hours, and RNA was extracted for transcriptome sequencing. GO and KEGG pathway analysis revealed that differentially expressed genes were enriched in the serine / threonine / tyrosine kinase pathway. Combined with TPP experiments, it was suggested that EphB3 may be a potential target of oligomycin A.

[0051] 3. Results

[0052] Experimental results are as follows Figure 3As shown, TPP and transcriptome data consistently support EphB3 as a direct target of oligomycin A.

[0053] III. Molecular computation techniques for predicting the binding site of oligomycin A to EphB3

[0054] To further analyze the binding site between oligomycin A and EphB3, the binding energies of the ligand binding site, phosphorylation site, and ATP binding site of oligomycin A and EphB3 were analyzed through structural simulation, molecular docking, and molecular dynamics simulation. The results are as follows: Figure 4 As shown, the binding energy of the ligand binding site of oligomycin A to EphB3 was found to be optimal.

[0055] IV. Biological effects of EphB3-mediated loratinib resistance:

[0056] 1. Materials and reagents:

[0057] (1) H3122-LR cells;

[0058] (2) Oligomycin A;

[0059] (3) siRNA-EphB3;

[0060] (4) Related antibodies, Western Blot reagents, CCK8 and EdU kits.

[0061] 2. Methods and Steps

[0062] H3122-LR cells were transfected with siRNA-EphB3 (with si-NC as a control), or H3122 cells were transfected with EV, GFP-EphB3, and Myc-EphB3 plasmids. Cells were then treated with loratinib ± oligomycin A, and CCK8, EdU, and cell colony formation assays were performed to analyze the effect of EphB3 on loratinib resistance.

[0063] 3. Results

[0064] Experimental results are as follows Figure 5 As shown, the results indicate that CCK8, EdU, and cell colony formation assays revealed that EphB3 mediates loratinib resistance, confirming the function of oligomycin A in reversing resistance through EphB3.

[0065] V. In vivo cellular experiments of oligomycin A reversing loratinib resistance

[0066] 1. Materials and reagents:

[0067] (1) BALB / c nude mice, 4-6 weeks old, male;

[0068] (2) H3122-LR cells;

[0069] (3) Loratinib (5 mg / kg / day, orally via gavage);

[0070] (4) Oligomycin A (0.3 mg / kg / d, intraperitoneal injection).

[0071] 2. Method and steps:

[0072] Operating steps: 3×10 6 H3122-LR cells were subcutaneously injected into the left hind limb of male BALB / c nude mice (4–6 weeks old). Once the tumor volume reached approximately 50 mm³, the mice were randomly divided into four groups (PBS control group, lorlatinib group, oligomycin A group, and combination therapy group). Treatment continued for 14 days (lorlatinib was administered by gavage at 5 mg / kg / day, and oligomycin A was administered intraperitoneally at 0.3 mg / kg / day). Tumor volume and body weight changes were measured and recorded every 3 days. After 14 days, the mice were sacrificed under excessive anesthesia, and tumor weight and volume were assessed to determine the biological effect of oligomycin A in reversing lorlatinib resistance in vivo, providing a reference for clinical translation.

[0073] Detection indicators: Changes in tumor volume and weight.

[0074] 3. Results:

[0075] Some experimental results are as follows Figure 6 As shown, the experimental results indicate that:

[0076] (1) Tumor growth was significantly inhibited in the combined drug group, and the tumor volume and weight were significantly smaller than those of the single drug and the control group.

[0077] (2) No significant weight loss indicates that the combination therapy is safe.

[0078] The above experimental results demonstrate that the oligomycin A combined with loratinib treatment regimen provided by this invention can effectively reverse ALK-TKI resistance, with a clear mechanism and good safety and clinical application potential. Those skilled in the art can replicate the effects of this invention based on the above steps and conventional experimental techniques, without requiring additional inventive effort.

[0079] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. The use of oligomycin A in the preparation of a drug for reversing resistance of tumor cells to loratinib, characterized in that, The tumor cells are non-small cell lung cancer cells.

2. A pharmaceutical composition for treating ALK-positive non-small cell lung cancer resistant to loratinib, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of oligomycin A and a therapeutically effective amount of loratinib.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is prepared as a dosage form for simultaneous, separate, or sequential administration.

4. The pharmaceutical composition according to claim 2 or 3, characterized in that, The oligomycin A and loratinib are prepared as single-unit dosage forms or as separate dosage forms.

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