Use of a benzimidazole-naphthol small molecule compound targeting ALDH1A1-K419 lactic acid modification in preparation of a drug for reversing EGFR-TKI drug resistance

Abstract

The application discloses application of a benzimidazole-naphthol small-molecule compound targeting ALDH1A1-K419 lactic acid modification in preparation of a drug for reversing EGFR-TKI drug resistance. Through research on an EGFR-TKI drug resistance related mechanism, a lead compound C-16 is screened and obtained, and the action of the lead compound is verified through molecular docking, surface plasmon resonance (SPR), cell activity experiments and an animal model, and results show that the compound exhibits certain anti-drug resistance activity in an in-vitro and in-vivo model, the compound can act on an ALDH1A1 related metabolic regulation process, and affect abnormal activation of a retinoic acid (RA) signal path, so that drug sensitivity of EGFR-TKI drug resistant tumor cells is improved. The application provides a new candidate compound and research basis for intervention of EGFR-TKI drug resistance related non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of a benzimidazole-naphthol small molecule compound targeting ALDH1A1-K419 lactation modification in the preparation of drugs to reverse EGFR-TKI resistance. Background Technology

[0002] Treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) has made significant progress since the clinical application of third-generation EGFR-tyrosine kinase inhibitors (TKIs, such as osimertinib (OSI)). However, the development of acquired resistance remains a core bottleneck limiting long-term patient survival, and there is an urgent need to elucidate novel molecular mechanisms underlying resistance in order to provide effective intervention strategies.

[0003] Tumor metabolic reprogramming, a key driver of drug resistance, is primarily manifested as abnormal glucose uptake and lactate accumulation induced by the Warburg effect. Lactylation, a newly discovered post-translational modification of metabolism-derived proteins, covalently modifies proteins to regulate their conformation, enzyme activity, or transcriptional function, becoming a crucial link between the metabolic microenvironment and altered tumor cell phenotypes.

[0004] aldehyde dehydrogenase 1A1 (ALDH1A1), as the rate-limiting enzyme catalyzing the conversion of retinal to retinoic acid (RA), is a key molecule in maintaining tumor stemness and mediating drug resistance. Current research largely focuses on the inhibition of ALDH1A1 enzymatic activity; however, whether ALDH1A1 is regulated by lactation modification in the complex tumor metabolic microenvironment, and how this post-translational modification drives EGFR-TKI resistance through aberrant activation of the RA signaling pathway, remains a research gap.

[0005] Therefore, in-depth research into the lactation modification mechanism of ALDH1A1 at specific sites and the development of candidate drugs that can specifically intervene in this target are of great theoretical significance and clinical application value for overcoming the problem of EGFR-TKI resistance. Summary of the Invention

[0006] Based on a systematic study of the ALDH1A1 lactation regulation axis, this invention screens and identifies a benzimidazole-naphthol small molecule compound that can target ALDH1A1-K419 lactation modification and reverse EGFR-TKI resistance, thus providing a new treatment option for clinically resistant patients.

[0007] According to a first aspect of the present invention, a benzimidazole-naphthol small molecule compound or a pharmaceutically acceptable salt / solvent / prodrug thereof is provided for use in the preparation of a medicament for overcoming resistance of EGFR-TKIs to EGFR-mutant tumors, the benzimidazole-naphthol small molecule compound being designated C-16 and having the molecular formula C 21 H 21 N3O, CAS number 364597-26-2, SMILES structure is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O.

[0008] As an optional implementation, the EGFR-TKI includes osimertinib.

[0009] As an optional implementation, the EGFR-mutated tumor includes EGFR-mutated non-small cell lung cancer.

[0010] As an alternative implementation, EGFR-TKI resistance can be overcome by targeting and inhibiting lactation modification at the K419 site of the ALDH1A1 protein and downregulating the retinoic acid RA-RARγ signaling pathway.

[0011] According to a second aspect of the present invention, a benzimidazole-naphthol small molecule compound or a pharmaceutically acceptable salt / solvent / prodrug thereof is provided for use in the preparation of a medicament that regulates the ALDH1A1-related metabolic pathway and inhibits ALDH1A1-K419 lactation modification, wherein the benzimidazole-naphthol small molecule compound is designated C-16 and has the molecular formula C 21 H 21 N3O, CAS number 364597-26-2, SMILES structure is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O.

[0012] As an optional implementation, C-16 reduces ALDH1A1 enzyme activity by inhibiting lactation modification at the ALDH1A1-K419 site, thereby regulating ALDH1A1-related metabolic pathways.

[0013] According to a third aspect of the invention, a pharmaceutical composition is provided comprising an effective amount of a benzimidazole-naphthol small molecule compound C-16 or a pharmaceutically acceptable salt / solvent / prodrug thereof, and a pharmaceutically acceptable carrier.

[0014] As an optional implementation, the pharmaceutical composition may further comprise an EGFR-TKI as a combination drug component.

[0015] As an optional implementation, the EGFR-TKI includes osimertinib.

[0016] As an optional implementation, the pharmaceutical composition may be a tablet, capsule, injection, suspension, or granule.

[0017] The present invention has the following significant beneficial effects:

[0018] This invention is the first to demonstrate that lactation modification at the K419 site of the ALDH1A1 protein is a key regulatory node driving osimertinib resistance in non-small cell lung cancer cells. By elucidating the novel metabolic-epigenetic regulatory axis of "ALDH1A1-K419 lactation-retinoic acid (RA)-RARγ", it fills the gap in the field of tumor drug resistance metabolic mechanisms and provides a precise molecular target for subsequent drug development targeting this pathway.

[0019] The benzimidazole-naphthol small molecule compound C-16 obtained by screening in this invention has been verified by molecular docking, surface plasmon resonance (SPR) and molecular dynamics simulations, which confirmed that it can specifically recognize and bind to the ALDH1A1-K419 lactation-related structural region, thereby effectively blocking this pathological modification and showing excellent targeted intervention potential.

[0020] Experimental results show that C-16, when used in combination with osimertinib, produces a significant synergistic anti-tumor effect. In in vitro cell experiments, C-16 effectively inhibited the abnormal proliferation of drug-resistant cells; in animal tumor models, C-16 significantly slowed the growth process of drug-resistant tumors. This demonstrates the clinical translational potential of this compound in restoring the sensitivity of tumor cells to EGFR-TKI drugs, providing a highly effective combination therapy strategy for clinically resistant patients.

[0021] In vivo experiments showed that C-16, at doses that exert antitumor activity, did not cause significant pathological damage to major organs such as the liver and kidneys. Good tolerability indicates that this compound not only has a clear therapeutic mechanism but also a wide safe therapeutic window, laying a solid foundation for its future development into a new clinical anticancer drug. Attached Figure Description

[0022] Figures 1-2 This is the result of lactation metabolomics sequencing analysis provided in the embodiments of the present invention; showing differential genes in lactation modification of lung cancer organoids from TKI-sensitive and drug-resistant patients ( Figure 1 ), and the modification specifically occurs at lysine position 419 (K419) ( Figure 2 ).

[0023] Figure 3The clinical sample verification results provided in this embodiment of the invention show the difference in immunohistochemical staining results of ALDH1A1-K419la immunohistochemical staining of paraffin tissue sections from TKI-sensitive (Naive) and drug-resistant (Resistant) lung cancer patients.

[0024] Figures 4-5 These are the experimental results of organoid function tests in TKI-sensitive and TKI-resistant lung cancer patients provided in the embodiments of the present invention; wherein, Figure 4 The results of Calcein-AM / PI staining of organoids derived from TKI-sensitive patients under OSI treatment conditions were obtained after treatment with control group (NC), lactate treatment, and ALDH1A1 overexpression (OE-ALDH1A1). Figure 5 The Calcein-AM / PI staining results are obtained after treating organoids from TKI-resistant patients under conditions of ALDH1A1 functional inhibition or mutation, suggesting that ALDH1A1-K419 lactation is involved in the formation of EGFR-TKI resistance phenotype.

[0025] Figures 6-7 These are the functional verification results of the retinoic acid (RA) and RAR signaling pathways provided in the embodiments of the present invention; wherein, Figure 6 Bright-field morphology images and live / dead fluorescence staining images of OSI-sensitive lung cancer patients derived from OSI-sensitive lung cancer patients under different treatment conditions are provided in the embodiments of the present invention, showing that exogenous addition of retinoic acid can induce drug resistance-related phenotypes in OSI-sensitive organoids; Figure 7 Bright-field morphology and live / dead fluorescence staining images of organoids derived from OSI-resistant lung cancer patients, as provided in embodiments of the present invention, after treatment with OSI, OSI combined with RAR inhibitors BMS195614 or MM11253, show that RAR inhibitors BMS195614 and MM11253 can improve the response of drug-resistant organoids to OSI.

[0026] Figures 8-9 These are the experimental results of the animal model provided in the embodiments of the present invention; wherein, Figure 8 The images of xenografts in different treatment groups and the curves showing the change in tumor volume over time provided in the embodiments of the present invention demonstrate that retinoic acid treatment can weaken the tumor-suppressive effect of OSI. Figure 9 The images of xenografts and the curves showing the changes in tumor volume over time in different combination drug groups provided in the embodiments of the present invention show that the combination of RAR inhibitors can improve the treatment response of drug-resistant tumors to OSI, among which MM11253 is more effective than BMS195614.

[0027] Figures 10-13 These are the results of compound screening and validation targeting the lactation-related structural regions of ALDH1A1-K419 provided in the embodiments of the present invention; wherein, Figure 10 These are virtual filtering results; Figure 11 The results are from surface plasmon resonance analysis, specifically SPR detection of C-16 and ALDH1A1. K419la The combination of affinity, Figure 11 Part A in the figure shows the real-time binding-dissociation curves of C-16 at different concentrations. Figure 11 Part B in the figure is the steady-state affinity fitting curve, and the calculated KD value is 79.8 μM; Figure 12 The results of the activity and toxicity evaluation in the cell model, Figure 12 Part A in the figure represents the dose-response curve after C-16 treatment of the drug-resistant cell line HCC827R, IC50. 50 It is 11.84 μM. Figure 12 Part B in the figure shows the cytotoxicity curve of normal human bronchial epithelial cells after HBE treatment with C-16, TC 50 It is 49.45 μM; Figure 13 Molecular dynamics simulation analysis of the C-16-target protein complex and the chemical structure of C-16 were performed. Figure 13 Part A in the text consists of C-16 and ALDH1A1 K419la Combined with the two-dimensional interaction diagram of the pocket, it is shown that C-16 can bind to surrounding residues through hydrogen bonding, hydrophobic interactions, and π-π stacking. Figure 13 Part B of the data presents the RMSD analysis of the protein and ligands during a 100 ns molecular dynamics simulation, showing that the complex remains relatively stable after initial equilibrium. Figure 13 Part C in the equation represents C-16 and ALDH1A1 during molecular dynamics simulations. K419la Analysis of the Coulomb interaction energy between them showed an average Coulomb energy of −8.77 kcal / mol, indicating a favorable electrostatic interaction between them.

[0028] Figures 14-16 These are the analysis results of the animal model provided in the embodiments of the present invention; wherein, Figure 14 To restore the sensitivity of drug-resistant PDO to OSI in vivo, the combined use of C-16 and OSI significantly inhibited the growth of drug-resistant xenografts. Figure 14 Part A of the image shows representative photographs of tumors removed from mice in different treatment groups. Figure 14 Part B in the figure shows the growth curves of tumor volume changes over time in mice under different treatment groups. C-16 can inhibit tumor growth, and C-16 combined with OSI treatment showed the strongest anti-tumor effect. Figure 15 HE staining of liver and kidney tissues under experimental conditions showed no obvious pathological damage; Figure 16 Immunohistochemical staining of ALDH1A1-K419 in tumor tissues of each group showed that the staining intensity of the control group (Vehicle / NC) and the OSI group was higher than that of the C-16 group and the C-16 combined OSI group. Detailed Implementation

[0029] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0030] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0032] As used in this article, the term "EGFR-TKI" refers to epidermal growth factor receptor tyrosine kinase inhibitors, a class of targeted therapeutic drugs mainly used to inhibit the abnormal activation of the EGFR (epidermal growth factor receptor) signaling pathway, thereby blocking the proliferation and survival of tumor cells.

[0033] As used in this article, the term "drug resistance" refers to the phenomenon that tumor cells develop resistance to drugs that were originally effective, leading to a decrease or failure of treatment efficacy.

[0034] As used in this article, the term "lactic acidification" refers to the post-translational modification of lysine residues in proteins using lactic acid as a substrate, which plays an important role in tumor metabolic reprogramming and drug resistance.

[0035] As used in this article, the term "ALDH1A1" refers to A1, a member of the acetaldehyde dehydrogenase 1 family. It is a key metabolic enzyme that catalyzes the conversion of retinal to retinoic acid and is involved in processes such as tumor cell stemness maintenance, metastasis, and drug resistance.

[0036] As used in this article, the term "ALDH1A1" K419la "ALDH1A1-K419 lactation modification" refers to the lactation modification of the lysine residue at position 419 of the ALDH1A1 protein. This invention is the first to discover that this modification is specifically upregulated in osimertinib-resistant samples.

[0037] As used in this article, the term "retinoic acid" or "RA" refers to a lipid-soluble signaling molecule generated by the conversion of retinal catalyzed by ALDH1A1, which regulates the transcription of downstream genes by binding to retinoic acid receptors.

[0038] As used in this article, the term "RAR" refers to retinoic acid receptors, including subtypes such as RARα, RARβ, and RARγ, which activate downstream signaling pathways after binding to retinoic acid.

[0039] As used in this article, the term "PDO" refers to patient-derived organoids, which are miniature tumor models formed by three-dimensional culture of patient tumor tissue in vitro, and which can retain the molecular characteristics and drug responsiveness of the original tumor.

[0040] As used in this article, the term "HCC827" refers to a human lung adenocarcinoma cell line carrying an EGFR mutation (E746-A750 deletion) that is sensitive to osimertinib.

[0041] As used in this article, the term "HCC827R" refers to a drug-resistant subline of HCC827 cells obtained through long-term osimertinib induction, which exhibits acquired resistance to osimertinib.

[0042] As used in this article, the term "IC" 50 "Hardest effective concentration" refers to the drug concentration required to inhibit 50% of cell proliferation.

[0043] As used in this article, the term "TC" 50 "Hydrotoxic concentration" refers to the drug concentration required to cause the death of 50% of normal cells.

[0044] As used in this article, the term "SPR" refers to surface plasmon resonance technology, which is used to monitor interactions between biomolecules in real time and to determine the binding affinity of compounds to target proteins.

[0045] As used in this article, the term "molecular docking" refers to a structure-based computer simulation technique used to predict the binding patterns and affinity of small molecule compounds to target proteins.

[0046] As used in this article, the term "virtual screening" refers to a method that uses computer technology to rapidly screen candidate molecules from a compound library that have potential binding activity to a target.

[0047] The results of this invention show that in EGFR-TKI resistant lung adenocarcinoma cells, GLUT3 expression is upregulated. The glucose uptake it drives provides a substrate for glycolysis, leading to intracellular lactate accumulation, which in turn promotes the increase of ALDH1A1-K419 lactation level and activates the downstream retinoic acid metabolic pathway, ultimately promoting the drug resistance phenotype.

[0048] Based on the above mechanism, this invention conducted compound screening targeting the lactation-related structural regions of ALDH1A1-K419, and obtained candidate compounds that can improve osimertinib (OSI) resistance phenotype.

[0049] In one embodiment of the present invention, the use of a benzimidazole-naphthol small molecule compound or a pharmaceutically acceptable salt / solvent / prodrug thereof is provided in the preparation of a medicament for overcoming resistance of EGFR-TKIs to EGFR-mutant tumors, wherein the benzimidazole-naphthol small molecule compound is designated C-16 and has the molecular formula C 21 H 21 N3O, CAS number 364597-26-2, SMILES structural formula is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O; wherein, the chemical structural formula of C-16 is shown in Formula I:

[0050] Formula I.

[0051] In some embodiments, EGFR-TKIs include osimertinib.

[0052] In some embodiments, EGFR-mutated tumors include EGFR-mutated non-small cell lung cancer.

[0053] In some embodiments, EGFR-TKI resistance is overcome by targeting and inhibiting lactation modification at the K419 site of the ALDH1A1 protein and downregulating the retinoic acid RA-RARγ signaling pathway.

[0054] In one embodiment of the present invention, the use of a benzimidazole-naphthol small molecule compound or its pharmaceutically acceptable salt / solvent / prodrug in the preparation of a drug that regulates the ALDH1A1-related metabolic pathway and inhibits ALDH1A1-K419 lactation modification is provided. The benzimidazole-naphthol small molecule compound is designated C-16 and has the molecular formula C0. 21 H 21 N3O, CAS number 364597-26-2, SMILES structure is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O.

[0055] In some embodiments, C-16 reduces ALDH1A1 enzyme activity by inhibiting lactation modification at the ALDH1A1-K419 site, thereby regulating ALDH1A1-related metabolic pathways.

[0056] In other embodiments of the present invention, a pharmaceutical composition is also provided, comprising an effective amount of a benzimidazole-naphthol small molecule compound C-16 or a pharmaceutically acceptable salt / solvent / prodrug thereof, and a pharmaceutically acceptable carrier.

[0057] In some embodiments, the pharmaceutical composition further comprises EGFR-TKI as a combination drug component.

[0058] In some embodiments, EGFR-TKIs include osimertinib.

[0059] In some embodiments, the pharmaceutical composition is a tablet, capsule, injection, suspension, or granule.

[0060] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0061] Unless otherwise specified, the following embodiments are all conventional methods.

[0062] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0063] Example 1

[0064] To investigate the metabolic modification basis of osimertinib (OSI) resistance in EGFR-mutant non-small cell lung cancer, lactation modification proteomics analysis was performed on organoids (PDOs) derived from 3 TKI-sensitive (naive) and 3 TKI-resistant patients. The TKI-sensitive group consisted of samples from patients sensitive to EGFR-TKI treatment, while the TKI-resistant group consisted of samples from patients who developed acquired resistance after EGFR-TKI treatment. After expansion under the same culture conditions, samples from both groups were collected for protein lactation modification proteomics analysis.

[0065] like Figure 1 As shown, compared with the TKI-sensitive group, the TKI-resistant group organoids showed extensive differential protein upregulation of lactation modification, among which the metabolic enzyme ALDH1A1 was a significantly highly lactated protein.

[0066] like Figure 2 As shown, further site analysis revealed that this modification specifically occurs at lysine 419 (K419) of the ALDH1A1 protein.

[0067] To validate this finding at the clinical level, paraffin-embedded tissue sections of lung cancer from TKI-sensitive and TKI-resistant patients were collected, and specific antibodies were prepared and immunohistochemically (IHC) stained.

[0068] like Figure 3 As shown, ALDH1A1 in tissue sections of drug-resistant patients K419la The expression level of this target was significantly higher than that of TKI-sensitive patients, confirming that this modified target has important indicative significance in clinically acquired drug resistance.

[0069] Example 2

[0070] To clarify ALDH1A1K419la To investigate the specific function in drug resistance, this embodiment conducted in vitro intervention experiments on TKI-sensitive and TKI-resistant organoids, and used Calcein-AM / PI cell viability staining to evaluate the survival and death of organoids after different treatments.

[0071] In organoids derived from TKI-sensitive patients, control group (NC), lactate group (LAC), ALDH1A1 overexpression group, and ALDH1A1 overexpression + lactate group were set up, and OSI treatment was added to each group.

[0072] The results are as follows Figure 4 As shown, OSI treatment can induce TKI-sensitive organoid death; exogenous lactate addition reduces organoid death; and the anti-apoptotic ability of organoids is further enhanced after overexpression of ALDH1A1 and combined lactate treatment, suggesting that lactate and ALDH1A1 jointly promote the formation of OSI resistance-related phenotypes.

[0073] In organoids derived from TKI-resistant patients, ALDH1A1 was knocked out, and control (WT), OSI, ALDH1A1-K419R complementation, and OSI+ALDH1A1-K419R complementation groups were set up; among them, ALDH1A1-K419R is a mutant that simulates the K419 site and cannot undergo lactation modification.

[0074] The results are as follows Figure 5 As shown, knocking out ALDH1A1 restored the sensitivity of drug-resistant organoids to OSI, but increased cell death. After reintroducing the ALDH1A1-K419R mutant, the survival ability of organoids under OSI treatment could not be effectively restored. This indicates that lactation modification at the ALDH1A1-K419 site is an important step in maintaining the OSI resistance phenotype.

[0075] The above results fully demonstrate that ALDH1A1 in lung cancer cells K419la The regulation of intracellular lactate levels is a core factor driving OSI drug resistance.

[0076] Example 3

[0077] Given that ALDH1A1 is a key enzyme catalyzing the conversion of retinaldehyde to retinoic acid (RA), the drug resistance mechanism in the downstream pathway was further verified.

[0078] First, organoids from TKI-sensitive patients were divided into three groups: NC, OSI, and OSI+RA. The NC group received no OSI treatment, the OSI group received osimertinib treatment, and the OSI+RA group received exogenous retinoic acid in addition to OSI treatment. After treatment, the bright-field morphology of the organoids was observed, and the viability of the organoids was detected using Calcein-AM / PI live / dead cell fluorescence staining.

[0079] like Figure 6 As shown, organoid death increased in the OSI group and decreased in the OSI+RA group, suggesting that exogenous RA addition can weaken the killing effect of OSI on sensitive organoids and induce drug resistance-related phenotypes.

[0080] Subsequently, in organoids derived from TKI-resistant patients, four groups were established: NC group, OSI group, OSI+BMS195614 group, and OSI+MM11253 group. The NC group did not receive OSI treatment, the OSI group received osimertinib treatment, the OSI+BMS195614 group received OSI treatment in addition to the RARα inhibitor BMS195614, and the OSI+MM11253 group received OSI treatment in addition to the RARγ inhibitor MM11253.

[0081] The results are as follows Figure 7 As shown, OSI treatment alone has limited killing effect on drug-resistant organoids; when combined with RAR inhibitors, the response of drug-resistant organoids to OSI is enhanced, with MM11253 combined with OSI treatment showing more significant cell death, suggesting that blocking RARγ signaling can more effectively restore the sensitivity of drug-resistant organoids to OSI.

[0082] To further validate the effect of the RA / RAR signaling pathway on the therapeutic effect of OSI in a nude mouse subcutaneous xenograft model. For the OSI-sensitive xenograft model, three groups were set up: Vehicle group, OSI group, and OSI+RA group. The Vehicle group received solvent control treatment, the OSI group received osimertinib treatment, and the OSI+RA group received osimertinib treatment in addition to retinoic acid treatment.

[0083] like Figure 8 As shown, tumor growth was significantly inhibited in the OSI group, while the tumor inhibition effect was weakened in the OSI+RA group, indicating that retinoic acid treatment can weaken the anti-tumor effect of OSI.

[0084] For the OSI-resistant xenograft model, four groups were set up: Vehicle group, OSI group, OSI+BMS195614 group, and OSI+MM11253 group. The Vehicle group was given solvent control treatment, the OSI group was given osimertinib treatment, the OSI+BMS195614 group was given osimertinib plus BMS195614 treatment, and the OSI+MM11253 group was given osimertinib plus MM11253 treatment.

[0085] like Figure 9 As shown, OSI treatment alone has limited inhibitory effect on the growth of drug-resistant tumors; however, tumor growth is inhibited when combined with a RAR inhibitor, with the OSI+MM11253 group showing the most significant inhibitory effect. This suggests that MM11253 combined with OSI can more effectively improve the treatment response of drug-resistant tumors to OSI, confirming that specific blocking of RARγ signaling is an effective way to reverse drug resistance.

[0086] Example 4

[0087] To obtain small molecule compounds that can target the lactation-related structures of ALDH1A1-K419, the Lys419 site was modeled based on the human ALDH1A1 crystal structure (PDB ID: 4WP7), and the Specs compound library was virtually screened using AutoDockVina.

[0088] like Figure 10 As shown, the molecular docking results indicate that candidate compound C-16 can enter the lactation-related binding pocket of ALDH1A1-K419 and form a good binding conformation with a docking binding energy of -9.7445 kcal / mol.

[0089] After screening and obtaining candidate molecules, their binding affinity to the target protein was verified using surface plasmon resonance (SPR) technology. The results are as follows: Figure 11 As shown, C-16 can stably bind to ALDH1A1 K419la ; and combined with cellular activity evaluation (IC) 50 With TC 50 Further screening was conducted, and the drug-resistant cell line HCC827R was treated with C-16 to detect IC50. 50 Human bronchial epithelial cells were treated with C-16 and HBE was used to detect TC. 50 ,like Figure 12 As shown, the C-16 has good effectiveness and safety.

[0090] Finally, candidate compound C-16 (C 21 H 21To evaluate its dynamic binding stability, a 100 ns all-atom molecular dynamics simulation of the C-16 and ALDH1A1-K419 lactated protein complex was performed using Desmond software under the TIP3P explicit water model and OPLS2005 force field. During the simulation, the protein backbone RMSD stabilized at approximately 2.0–2.6 Å after initial relaxation, and the ligand remained within the binding pocket throughout. The MM / GBSA calculation yielded an average binding free energy of -52.32 kcal / mol for the complex.

[0091] The above results indicate that C-16 can form a relatively stable complex with the lactation-related structural regions of ALDH1A1-K419 under simulated conditions. Figure 13 It also showed inhibitory potential in drug resistance-related models.

[0092] Example 5

[0093] To evaluate the in vivo effects of compound C-16, a drug-resistant PDO xenograft model was constructed in nude mice, and control (Vehicle), OSI group, C-16 group, and C-16 combined with OSI group were set up (OSI was administered orally by gavage at 5 mg / kg daily, C-16 was administered by tail vein injection at 10 mg / kg every other day, the combined group received both administration methods until the end of the experiment, and the control group was treated with an equal volume of physiological saline).

[0094] like Figure 14 As shown, both C-16 monotherapy and C-16 combined with OSI can inhibit the growth of drug-resistant tumors, with the combination therapy group showing a more significant inhibitory effect.

[0095] like Figure 15 As shown, HE staining of liver and kidney tissues from animals in group C-16 and group C-16 combined with OSI showed no obvious pathological damage, indicating that no obvious liver and kidney toxicity was observed under this administration condition.

[0096] Further immunohistochemical staining with ALDH1A1-K419 lactated stained tumor tissues from each group was performed, and three tissue sections from each group were taken for observation; Figure 16 As shown, the control group and OSI group showed stronger staining, while the C-16 group and the C-16 combined with OSI group showed weaker staining.

[0097] The above results indicate that C-16 has an inhibitory effect on the growth of drug-resistant tumors in animal models and can affect the lactation-related phenotype of ALDH1A1-K419.

[0098] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. The use of a benzimidazole-naphthol small molecule compound or a pharmaceutically acceptable salt / solvent / prodrug thereof in the preparation of a medicament for overcoming resistance to EGFR-TKIs in EGFR-mutant tumors, characterized in that, This benzimidazole-naphthol small molecule compound is designated C-16, with the molecular formula C0. 21 H 21 N3O, CAS number 364597-26-2, SMILES structure is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O.

2. The application according to claim 1, characterized in that, The EGFR-TKI includes osimertinib.

3. The application according to claim 1, characterized in that, The EGFR-mutated tumors include EGFR-mutated non-small cell lung cancer.

4. The application according to claim 1, characterized in that, By targeting and inhibiting lactation modification at the K419 site of the ALDH1A1 protein, the RA-RARγ signaling pathway of retinoic acid is downregulated to overcome EGFR-TKI resistance.

5. The use of a benzimidazole-naphthol small molecule compound or its pharmaceutically acceptable salt / solvent / prodrug in the preparation of a drug that regulates the ALDH1A1-related metabolic pathway and inhibits ALDH1A1-K419 lactation modification, characterized in that, This benzimidazole-naphthol small molecule compound is designated C-16, with the molecular formula C0. 21 H 21 N3O, CAS number 364597-26-2, SMILES structure is CCCN1C2=CC=CC=C2N=C1NCC3=C(C=CC4=CC=CC=C43)O.

6. The application according to claim 5, characterized in that, The C-16 reduces ALDH1A1 enzyme activity by inhibiting lactation modification at the ALDH1A1-K419 site, thereby regulating ALDH1A1-related metabolic pathways.

7. A pharmaceutical composition, characterized in that, It contains an effective amount of a benzimidazole-naphthol small molecule compound C-16 or its pharmaceutically acceptable salt / solvent / prodrug, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also contains EGFR-TKI as a combination drug component.

9. The pharmaceutical composition according to claim 8, characterized in that, The EGFR-TKI includes osimertinib.

10. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, injections, suspensions, or granules.

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