Application of lumepirozol tosylate in the preparation of drugs for treating tumors
Through molecular docking and screening methods, it was discovered that rumeperone tosylate has inhibitory activity against HAT1, which solves the problem of developing HAT1 targeted inhibitors in the existing technology, realizes effective treatment of a variety of solid tumors, and broadens its clinical application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN TUMOR HOSPITAL
- Filing Date
- 2026-06-03
- Publication Date
- 2026-06-30
AI Technical Summary
The development of HAT1-targeting inhibitors in the current technology faces challenges, especially the lack of efficient screening processes and reports of in vitro and in vivo antitumor active compounds. Furthermore, the known selective inhibitors are limited and difficult to effectively inhibit various solid tumors such as liver cancer, melanoma, and colorectal cancer.
Using lumepirozoline tosylate as a HAT1-targeting compound, candidate drugs with in vitro and in vivo activities were screened through molecular docking, MM-GBSA binding free energy screening, and protein-ligand interaction fingerprinting analysis. The dosage was 40 mg/kg, which was used to prepare drugs for the treatment of solid tumors with high HAT1 expression, such as liver cancer, melanoma, and colorectal cancer.
This study expands the clinical application of rumepiride tosylate, providing a new drug option for cancer treatment. It significantly inhibits the growth of liver cancer, melanoma, and colorectal cancer, and enhances the anti-tumor effect when used in combination with PD-1 monoclonal antibodies.
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Figure CN122297474A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the use of lumepirozol tosylate in the preparation of drugs for treating tumors. Background Technology
[0002] Histone acetyltransferase 1 (HAT1) is a key enzyme catalyzing the acetylation of histone H4 lysine residues, playing a crucial role in chromatin structure regulation, gene expression, and cell cycle progression. Recent studies have shown that HAT1 is abnormally highly expressed in various malignant tumors, including liver cancer, colorectal cancer, and breast cancer, and its expression level is significantly correlated with tumor proliferation, invasion, and poor prognosis. Targeting and inhibiting HAT1 activity with small molecule inhibitors has emerged as a potential novel anti-tumor therapeutic strategy.
[0003] However, the discovery of HAT1-targeted inhibitors faces significant challenges. First, drug development research targeting HAT1 is still in its early stages, with a very limited number of known selective inhibitors possessing clearly defined activity. Second, high-throughput virtual screening targeting the HAT1 protein structure lacks a mature and efficient standardized process. Although computational chemistry methods such as molecular docking and binding free energy calculations have been widely applied in drug discovery, establishing a screening process that effectively enriches active lead compounds based on the structural characteristics of HAT1 (such as the potential presence of multiple allosteric binding sites and active pocket properties) remains a critical technical problem to be solved in this field. This process should include appropriate receptor preparation strategies, docking parameter settings, evaluation criteria, and post-processing analysis methods.
[0004] Furthermore, there are no reported cases in the existing technology of HAT1 inhibitors obtained through computational screening methods that have broad-spectrum anti-tumor activity in vitro and in vivo, especially candidate compounds that can simultaneously inhibit the growth of multiple solid tumors such as liver cancer, melanoma, and colorectal cancer.
[0005] Lumateperone (molecular formula C) 24 H 28 FN3O (molecular weight 393.5 g / mol) is an atypical antipsychotic drug primarily used to treat depressive episodes associated with schizophrenia and bipolar disorder. It improves symptoms by modulating dopamine and serotonin receptor activity and was officially introduced to the Chinese market in 2024. Lumateperone tosylate (molecular formula C...) is another drug used in this study. 31 H 36FN3O4S (molecular weight 565.699 g / mol) are different forms of the same drug; the former is the generic name, and the latter is the specific salt form. Their pharmacological effects and clinical applications are basically the same, but the tosylate form is more conducive to drug stability and absorption. However, whether rumepiride tosylate has HAT1 inhibitory activity and its potential application in tumor treatment has not been reported.
[0006] Therefore, this invention is proposed. Summary of the Invention
[0007] To solve the above-mentioned technical problems, the present invention provides rumeperone toluenesulfonate (molecular formula C... 31 H 36 Application of FN3O4S (molecular weight 565.699 g / mol) in the preparation of drugs for treating tumors. A highly efficient, accurate, and targeted method for screening HAT1-targeting compounds was developed, leading to the discovery of candidate drugs with clear in vitro and in vivo activities. Simultaneously, the clinical application of lumepirozoline tosylate was broadened, providing new drug options for tumor treatment.
[0008] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides the use of lumepirozol tosylate in the preparation of medicaments for treating tumors.
[0009] Furthermore, the tumor is a solid tumor.
[0010] Furthermore, the tumor is a tumor that highly expresses HAT1.
[0011] Furthermore, the tumors include: liver cancer, melanoma, and colorectal cancer.
[0012] Furthermore, the liver cancer is HepG2 liver cancer, the melanoma is B16 melanoma, and the colorectal cancer is MC38 colorectal cancer.
[0013] Furthermore, the rumepiride tosylate or a pharmaceutically acceptable dose thereof is 40 mg / kg.
[0014] Furthermore, the screening method for rumepiride tosylate includes the following steps: S1. Using molecular docking, the small molecules in the compound library are docked and scored at least one binding site of the HAT1 protein. S2. Select compounds that meet the following criteria: docking score ≤ -8, binding free energy (MM-GBSA) calculated by molecular mechanics / generalized Born surface area method ≤ -30 kcal / mol, and ligand strain energy ≤ 10 kcal / mol. S3. Perform protein-ligand interaction fingerprint analysis on the compounds screened in S2; S4. Perform cluster analysis on the analyzed compounds, set a similarity threshold of ≥85%, and select the compound with the optimal binding free energy obtained from molecular mechanics calculations in each class as candidate molecules.
[0015] Furthermore, the binding sites include: small molecule binding sites in the crystal structure or sites predicted by AlphaFold; Alternatively, it could be a combination of small molecule binding sites in the crystal structure and sites predicted by AlphaFold.
[0016] Furthermore, in S3, the amino acid residues that interact frequently with the ligand in the protein-ligand interaction fingerprint analysis include at least one of Gly251, Gly253, Met241, Asp277, Ser279, and Ser281.
[0017] The present invention also provides a pharmaceutical composition comprising the above-mentioned lumepirozol tosylate and a pharmaceutically acceptable carrier or excipient.
[0018] Furthermore, the dosage form of the pharmaceutical composition includes any one of the following: injection, tablet, capsule, or oral liquid.
[0019] The present invention has the following technical effects: This invention provides a novel use of lumepirozon tosylate in the preparation of drugs for treating tumors. As a known drug, lumepirozon tosylate demonstrates antitumor activity, particularly showing good inhibitory effects against solid tumors such as liver cancer, melanoma, and colorectal cancer. Through molecular docking and virtual screening methods, its potential mechanism of action on the HAT1 protein was determined, and an effective dosage (e.g., 40 mg / kg) was optimized. This not only expands the clinical application range of lumepirozon tosylate and provides a new drug option for tumor treatment, but also lays a scientific foundation for its further development into a targeted antitumor drug based on a clearly defined molecular target and screening method, possessing significant translational medicine value. Attached Figure Description
[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0021] Figure 1 The molecular structural formula of lumepirocin is C10. 24H 28 FN3O; Figure 2 The molecular structural formula of lumepirobentosulfonate is C10. 31 H 36 FN3O4S; Figure 3 Molecular docking diagram of lumepirozoline tosylate and HAT1; Figure 4 Experimental results showing that rumepiride toluene inhibits the growth of Huh7 liver cancer cells; Figure 5 Cell viability was detected using the CCK-8 assay kit. A) investigated the effects of lumepirozoline tosylate on human HepG2 liver cancer cells and HAT1 knockout human HepG2 liver cancer cells; B) examined the efficacy of lumepirozoline tosylate on mouse human Hepa1-6 liver cancer cells and HAT1 knockout Hepa1-6 cells. Figure 6 The experimental results of lumepirozol tosylate inhibiting liver cancer growth in C57BL / 6 mice are shown. A represents the liver cancer growth curve; B represents a tumor photograph; C represents tumor weight; and D represents the survival curve. (Student's best test) P <0.05, P <0.01, P <0.001; Figure 7 Results of experiments on the inhibition of melanoma growth by lumepirozol tosylate in C57BL / 6 mice. A shows the melanoma growth curve; B shows a tumor photograph; C shows the tumor weight; Student's best test... P <0.05, P <0.01, P <0.001; Figure 8 Lumepiride tosylate inhibited colorectal cancer growth in C57BL / 6 mice. A represents the colorectal cancer growth curve; B represents a tumor photograph; C represents tumor weight; Student's best test... P <0.05, P <0.01, P <0.001; Figure 9Experimental results on the antitumor effect of rumepiride to sensitize anti-PD-1 monoclonal antibody, where A is the liver cancer growth curve; B is a tumor photograph; C is the tumor weight; Student's best test. P <0.05, P <0.01, P <0.001. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0023] In a first aspect, the present invention provides the use of lumepirozoline tosylate in the preparation of medicaments for treating tumors.
[0024] In some embodiments, the tumor is a solid tumor.
[0025] In some embodiments, the tumor is a tumor that highly expresses HAT1.
[0026] In some embodiments, the tumor includes liver cancer, melanoma, and colorectal cancer.
[0027] In some embodiments, the liver cancer is HepG2 liver cancer, the melanoma is B16 melanoma, and the colorectal cancer is MC38 colorectal cancer.
[0028] In some embodiments, the lumepirozoline tosylate or a pharmaceutically acceptable dose thereof is 40 mg / kg.
[0029] In some embodiments, the screening method for rumepiride tosylate includes the following steps: S1. Using molecular docking, the small molecules in the compound library are docked and scored at least one binding site of the HAT1 protein. S2. Select compounds that meet the following criteria: docking score ≤ -8, binding free energy (MM-GBSA) ≤ -30 kcal / mol calculated by molecular mechanics / generalized Born surface area method, and ligand strain energy ≤ 10 kcal / mol. S3. Perform protein-ligand interaction fingerprint analysis on the compounds screened in S2; S4. Perform cluster analysis on the analyzed compounds, set a similarity threshold of ≥85%, and select the compound with the optimal binding free energy obtained from molecular mechanics calculations in each class as candidate molecules.
[0030] In some embodiments, the binding sites include: small molecule binding sites in the crystal structure or sites predicted by AlphaFold; Alternatively, it could be a combination of small molecule binding sites in the crystal structure and sites predicted by AlphaFold.
[0031] In some embodiments, the amino acid residues that interact frequently with the ligand in the protein-ligand interaction fingerprint analysis include at least one of Gly251, Gly253, Met241, Asp277, Ser279, and Ser281.
[0032] In a second aspect, the present invention also provides a pharmaceutical composition comprising the above-mentioned lumepirozoline tosylate and a pharmaceutically acceptable carrier or excipient.
[0033] In some embodiments, the dosage form of the pharmaceutical composition includes any one of injection, tablet, capsule, or oral liquid.
[0034] The following is a detailed explanation using specific embodiments: Example 1 1.1.1 Preparation of the compound library: The compound library selected for this virtual screening was L1000. During the screening process, the compound library was first processed using the LigPrep module in the Schrödinger software, with the forcefield set to OPLS4. The Epik method was used to protonate and desalt the compound library at pH 7.0±2.0 to generate tautomers while maintaining the original atomic chirality.
[0035] 1.1.2 Protein preparation: The Protein Preparation Wizard module in Schrödinger software was used to perform bond order optimization, hydrogenation, disulfide bond allocation, hydrogen bond allocation, and energy minimization on the protein. This screening selected three sites: a small molecule binding site based on the crystal structure and two sites predicted by AlphaFold. The Receptor Grid Generation module in Schrödinger software was used to calculate the properties of the docking pocket.
[0036] 1.1.3 Virtual Filtering: Import the receptor and ligand files prepared in steps 1.1.1 and 1.1.2. Use a two-step screening mode with progressively increasing precision using a step-wise strategy (SP [Standard Precision Mode] → MM-GBSA). Each step uses a small molecule flexible docking method. After docking, energy optimization is performed. Since the compound library is relatively small, SP precision is selected as the starting point, and 50% of the data is retained for the next step of analysis.
[0037] 1.2.1 Affinity Analysis: A total of 2065 structures were output by molecular docking. When further analyzing the screened compounds, the focus was first on their scoring functions: docking scores ranged from -4 to -14, while standard precision mode scores ranged from -100 to 0. In this analysis, compounds with docking scores ≤ -8 and MM-GBSA ≤ -30 kcal / mol were selected as target compounds. To exclude compounds with conformational instability during binding, molecules with ligand strain energies ≤ 10 kcal / mol were screened for further study. The specific screening results are as follows: 176 compounds were obtained from site1; 27 compounds from site2; and 0 compounds from site3. A total of 203 compounds were screened. Given that the number of compounds from site1 is significantly greater than that from site2, compounds from site1 will be selected for further study.
[0038] 1.2.2 Protein-ligand interaction analysis (PLIF): The results were imported into the MOE for receptor-ligand interaction fingerprinting (PLIF) analysis to examine the amino acid residues that interact with the small molecule compounds. PLIF analysis showed that the high-frequency amino acids corresponding to site 1 were Gly251, Gly253, Met241, Asp277, Ser279, and Ser281. All of these compounds interacted with proteins.
[0039] 1.3 Cluster Analysis Cluster analysis was performed on the compounds obtained in the previous step. The cluster module of MOE software was used for cluster analysis, with MACCS fingerprint as the feature parameter and a similarity threshold of 85%. The molecules were ultimately clustered into 115 categories. For subsequent screening, the molecule with the best MM-GBSA performance was selected from each category, resulting in a total of 115 molecules. The molecular docking analysis results of lumepirozoline tosylate and HAT1 are shown below. Figure 3 As shown.
[0040] The molecular docking mode of lumepirozoline tosylate and HAT1 protein was analyzed using the MOE software: the docking fraction of lumepirozoline tosylate with HAT1 protein was -8.57, the MM-GBSA binding energy was -52.92 kJ / mol, hydrogen bonds were formed with protein binding sites G251 and S279, and CH-π interactions were formed with G253 and S279.
[0041] Example 2: Inhibitory effect of the HAT1-targeting drug lumepirozon tosylate on Huh7 hepatocellular carcinoma cells, HepG2 hepatocellular carcinoma cells, HAT1 knockout HepG2 hepatocellular carcinoma cells, Hepa1-6 mouse hepatocellular carcinoma cells, and HAT1 knockout Hepa1-6 hepatocellular carcinoma cells. After virtual screening, 115 candidate small molecule drugs were initially identified. Based on the binding score, the top 30 were selected, and relevant drug information was retrieved. Among them, 10 were identified as marketed drugs that were not intended for anticancer use. Therefore, the in vitro anticancer activity of these 10 non-anticancer drugs was systematically evaluated.
[0042] The cells were digested and collected to prepare a single-cell suspension. Cell counting was performed, and the cells were diluted to the desired density with complete culture medium, 5000 cells (100 μL) per well.
[0043] Experimental Groups: Blank control group: only 100 μL of complete culture medium (cell-free) was added for zeroing.
[0044] Control group: only cells and culture medium were added (no drug treatment), representing 100% cell viability.
[0045] Experimental group: Cells + culture medium + 20 μM of different test drugs (purchased from Shanghai Taoshu Technology Co., Ltd.), with at least 3 replicates per group.
[0046] Each group should have at least three replicates. Pre-incubate the culture plate at 37°C with 5% CO2 for 24 hours. Prepare the CCK8 working solution, calculating the required total volume by adding 10 μL of CCK8 stock solution to each well. Replace the culture medium with the CCK8 working solution, discard the culture plate, gently shake to mix, and return to the incubator for 3 hours in the dark. Use a microplate reader, setting the detection wavelength to 490 nm. Immediately measure the absorbance (OD) value of each well.
[0047] The absorbance results measured at 490 nm were as follows: the average absorbance of the control group was 0.4962. In the experimental groups: the average absorbance of the tetromod group was 0.4954, the average absorbance of the rumepiride tosylate group was 0.4018, the average absorbance of the clonazolidine group was 0.527, the average absorbance of the cefdinir group was 0.524, the average absorbance of the indobufen group was 0.4842, the average absorbance of the pseudomicillin group was 0.4858, the average absorbance of the thiamine pyrophosphate group was 0.5046, the average absorbance of the midaprazole hydrochloride group was 0.4774, the average absorbance of the cyclothiazide group was 0.4602, and the average absorbance of the mitabiva group was 0.422. After normalizing the absorbance values at 490 nm from the control group, the results represent the average cell activity of the rumepiride tosylate group relative to the control group. Student's first test, *P<0.05, **P<0.01, ***P<0.001, experimental results are as follows: Figure 4 As shown.
[0048] The results of the CCK-8 experiment showed that the antipsychotic drug rumepiride tosylate and the drug mitabiva for treating hemolytic anemia could inhibit the proliferation of human liver cancer cells Huh7, and rumepiride tosylate had a better inhibitory effect on liver cancer cells Huh7 than mitabiva.
[0049] Example 3: Tumor-bearing experiment in C57BL / 6 mice to detect the inhibitory effect of rumeperone tosylate on the growth of Hepa1-6 liver cancer cells, B16 melanoma cells, and MC38 colorectal cancer tumors in mice. C57BL / 6 mice aged 4-6 weeks were subcutaneously inoculated with Hepa1-6 cells, B16 cells, and MC38 cells to induce tumor formation (B16 1×10⁻⁶ cells). 6 / each, Hepa1-6 5×10 6 / each, MC38 1×10 6 / ani), and after 1 week, administer 40 mg / kg of lumepirozoline tosylate (purchased from Shanghai Taoshu Technology Co., Ltd.) by gavage, daily. Monitoring of its effects on tumors revealed that lumepirozoline tosylate significantly inhibited the growth of liver cancer, melanoma, and colorectal cancer in mice. Experimental results are as follows: Figures 5-8 As shown.
[0050] from Figure 5 It can be seen that the inhibition rate of lumepirozol tosylate on HAT1 knockout cells is significantly lower than that on human cells.
[0051] The mean absorbance of human hepatocellular carcinoma HepG2 cells untreated with rumepiride tosylate (control group) was 0.7684, while the mean absorbance after treatment was 0.6246. After normalizing the absorbance of the control group at 490 nm, the inhibition rate of rumepiride on human hepatocellular carcinoma HepG2 cells was calculated, and the mean inhibition efficiency was found to be 18.71%. Figure 5 A); The mean absorbance of HAT1 knockout human hepatocellular carcinoma HepG2 cells without treatment with rumepiride tosylate (control group) was 0.5546, and the mean absorbance after treatment was 0.4952. After normalizing the absorbance of the control group at 490 nm, the inhibition rate of rumepiride tosylate on HAT1 knockout human hepatocellular carcinoma HepG2 cells was calculated, and the mean inhibition efficiency was found to be 10.71%. Figure 5 B).
[0052] The average absorbance of human Hepa1-6 cells untreated with lumepirozol tosylate (control group) was 1.0735, while the average absorbance after treatment was 0.7822. After normalizing the absorbance at 490 nm in the control group, the inhibition rate of lumepirozol against human Hepa1-6 cells was calculated, showing an average inhibition efficiency of 27.14%. The average absorbance of HAT1 knockout Hepa1-6 cells untreated with lumepirozol tosylate (control group) was 1.3987, while the average absorbance after treatment was 1.2403. After normalizing the absorbance at 490 nm in the control group, the inhibition rate of lumepirozol tosylate against HAT1 knockout Hepa1-6 cells was calculated, showing an average inhibition efficiency of 11.32%. P <0.05, P <0.01, P <0.001.
[0053] from Figures 6-8 The results show that rumeperone tosylate can effectively inhibit the proliferation of liver cancer Hepa1-6 cells, melanoma B16 cells, and colorectal cancer MC38 cells.
[0054] Example 4: Detection of the sensitizing effect of rumepiride to antagonize the antitumor effect of PD-1 monoclonal antibody.
[0055] A subcutaneous hepatocellular carcinoma model was established in C57BL / 6 mice by implanting Hepa1-6 hepatocellular carcinoma cells onto the tumor. The tumors were allowed to grow to 80 mm in size. 3 When the tumor volume is around 1500 mmHg, administer anti-PD-1 monoclonal antibody (10 mg / kg, intraperitoneal injection, twice a week for two weeks) and rumepiride (oral gavage, 40 mg / kg, daily gavage). Measure the tumor volume every two days, and continue treatment until the largest tumor volume does not exceed 1500 mmHg.3 Mice were euthanized, and tumor tissue was photographed. The experimental results of the antitumor effect of rumeperone tosylate-enhanced anti-PD-1 monoclonal antibody (PD-1 monoclonal antibody sourced from Shanghai Taoshu Biotechnology Co., Ltd., catalog number T78269 (Anti-Mouse PD-1 Antibody (RMP1-14)) are as follows: Figure 9 As shown. From Figure 9 The results show that rumeperone tosylate can be used in combination with PD-1 to achieve therapeutic effects on liver cancer.
[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.
Claims
1. The application of lumepirozol tosylate in the preparation of drugs for treating tumors, characterized in that, The tumors include: liver cancer, melanoma, and colorectal cancer.
2. The use of lumepirozol tosylate according to claim 1 in the preparation of a medicament for treating tumors, characterized in that, The tosylate lumepirocin or its pharmaceutically acceptable dose is 40 mg / kg.
3. The use of lumepirozol tosylate according to any one of claims 1-2 in the preparation of a medicament for treating tumors, characterized in that, The screening method for rumepiride tosylate includes the following steps: S1. Using molecular docking, the small molecules in the compound library are docked and scored at least one binding site of the HAT1 protein. S2. Select compounds that meet the following criteria: docking score ≤ -8, binding free energy ≤ -30 kcal / mol obtained by molecular mechanics calculation and ligand strain energy ≤ 10 kcal / mol. S3. Perform protein-ligand interaction fingerprint analysis on the compounds screened in S2; S4. Perform cluster analysis on the analyzed compounds, set a similarity threshold of ≥85%, and select the compound with the optimal binding free energy obtained from molecular mechanics calculations in each class as candidate molecules.
4. The use of lumepirozol tosylate according to claim 3 in the preparation of a medicament for treating tumors, characterized in that, The binding sites include: small molecule binding sites in the crystal structure or sites predicted by AlphaFold; Alternatively, it could be a combination of small molecule binding sites in the crystal structure and sites predicted by AlphaFold.
5. The use of lumepirozol tosylate according to claim 3 in the preparation of a medicament for treating tumors, characterized in that, In S3, the amino acid residues that interact frequently with the ligand in the protein-ligand interaction fingerprint analysis include at least one of Gly251, Gly253, Met241, Asp277, Ser279, and Ser281.