Use of cinobufagin or its active ingredient combination in the preparation of an in vitro MCMs protein inhibitor for non-disease treatment purposes

Abstract

The application relates to application of cinobufagin or an active ingredient combination thereof in preparation of an in-vitro MCMs protein inhibitor for non-disease treatment purposes. The application can directly target MCMs protein by applying the cinobufagin or the active ingredient combination thereof in preparation of the in-vitro MCMs protein inhibitor, so as to realize accurate intervention on cell DNA replication. Experiments prove that the cinobufagin and the active ingredient combination thereof can significantly inhibit the activity of tumor cells such as liver cancer cells, but have no obvious influence on the activity of normal liver cells, indicating that the cinobufagin and the active ingredient combination thereof have selective inhibiting effect on fast proliferating cells (such as tumor cells), instead of killing all cells indiscriminately.

Description

Technical Field

[0001] This invention belongs to the field of biochemical reagents and protein interaction regulation reagents, specifically relating to the application of bufotin or a combination of its active ingredients in the preparation of in vitro MCMs protein inhibitors for non-disease treatment purposes. Background Technology

[0002] Minichromosome maintenance proteins (MCMs) are a family of proteins that play a central role in the initiation and elongation of DNA replication in eukaryotes. The MCM2-7 hexamer complex, as a core component of DNA replication helicase, is responsible for unwinding the DNA double helix to initiate the replication fork, a critical rate-limiting step in the cell cycle transition from G1 to S phase. Recent studies have shown that MCMs are abnormally highly expressed in various malignant tumor tissues, and their expression levels are significantly positively correlated with tumor malignancy, metastatic potential, and poor patient prognosis, making them a novel target for tumor biotherapy. Currently, some studies have attempted to use PROTAC technology to design small molecule drugs targeting MCMs, aiming to inhibit tumor growth by degrading MCM proteins. However, reports on MCM inhibitors remain very limited, and globally, there is a lack of compounds that can directly target MCMs and have entered clinical trials.

[0003] Bufotalin is a refined extract from the dried skin of the Chinese giant toad or the black-rimmed toad (Bufo gargarizans). It mainly contains bufotalin lactones such as bufotalin, bufotalin, and bufotalinol, as well as indole alkaloids. In recent years, scholars both domestically and internationally have conducted extensive research on the antitumor mechanism of bufotalin, revealing its multi-target and multi-pathway regulatory characteristics, such as exerting antitumor effects by regulating signaling pathways like PI3K / AKT, MAPK, and Wnt / β-catenin. However, its direct intracellular protein targets are not fully elucidated, and there are no reports of bufotalin directly targeting the core DNA replication proteins MCMs. Currently, the direct interaction between bufotalin and MCMs proteins and its impact on MCM function remains a blank area. Summary of the Invention

[0004] This invention provides the application of bufotoxin or a combination of its active ingredients in the preparation of in vitro MCMs protein inhibitors for non-disease treatment purposes, aiming to reveal a novel molecular mechanism of bufotoxin's anti-tumor activity and to provide new lead compounds and theoretical basis for the development of anti-tumor drugs or research reagents targeting MCMs.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] Application of bufotin or a combination of its active ingredients in the preparation of in vitro MCMs protein inhibitors for non-disease therapeutic purposes. By applying bufotin or a combination of its active ingredients to the preparation of in vitro MCMs protein inhibitors, MCMs proteins can be directly inhibited, thereby achieving precise intervention in cellular DNA replication.

[0007] Preferably, the MCMs proteins include at least one of MCM3, MCM5, or MCM7. By specifying the specific type of the MCMs proteins as MCM3, MCM5, or MCM7, the inhibitory effect of bufotoxin and its active ingredient combination can be exerted more precisely, because experimental data show that bufotoxin has the most significant degradation effect on these three proteins.

[0008] Preferably, the in vitro MCMs protein inhibitor is an MCMs protein degrader; the application specifically involves: promoting the degradation of MCMs proteins via the ubiquitin-proteasome pathway by inhibiting the interaction between MCMs proteins and their molecular chaperone MCMBP proteins. This inhibition of protein-protein interactions (PPIs) effectively disrupts the stability of the MCMs complex, thereby promoting MCMs protein degradation and ultimately inhibiting the initiation and elongation of DNA replication.

[0009] Preferably, the active ingredient combination of the bufalin is a bufotalin compound, which is composed of 11 monomeric compounds including bufalin, bufofagin, bufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, areonobufagin, gamabufotalin, marinobufagin, and resibufagin.

[0010] Preferably, the molar ratio of each monomer compound in the active ingredient combination is 1:1:1:1:1:1:1:1:1:1:1:1.

[0011] Preferably, the active ingredient combination competitively blocks the interaction between MCMs and MCMBP in vitro by acting on different amino acid residues of the MCMBP protein. This mechanism of directly binding to and competitively blocking the MCMBP protein efficiently inhibits the MCMBP-MCMs interaction, achieving a targeted inhibitory effect in in vitro biochemical experiments. This active ingredient combination can serve as a small molecule inhibitor targeting the MCMBP-MCMs protein interaction interface. This mode of action is revealed for the first time in this invention. Existing technologies, the antitumor mechanisms of bufotoxin and its active ingredients mainly involve inducing apoptosis, regulating immunity, and reversing multidrug resistance; there are no reports on targeting the MCMBP-MCMs interaction.

[0012] Preferably, the in vitro MCMs protein inhibitor is a research reagent or in vitro biochemical assay kit used in cell biology research. By specifying the final industrial product form as a research reagent or kit, the needs of in vitro research and biochemical assays can be met, demonstrating good practicality.

[0013] The application of a bufotoxin or a combination of its active ingredients in the preparation of a research reagent for reducing the expression level of MCM3 protein in in vitro cell experiments for non-disease treatment purposes, wherein the combination of active ingredients comprises bufotoxin, bufotoxin, bufotoxin ether, bufotoxin ether, deacetylated bufotoxin, bufotoxin ether, bufotoxin ether, bufotoxin ether, bufotoxin ether, bufotoxin ether, and bufotoxin ether.

[0014] Preferably, the research reagent is also used to reduce the expression levels of MCM5 and / or MCM7 proteins.

[0015] In the described application, the bufotoxin or the combination of active ingredients can increase the ubiquitination level of the MCM3 protein and reduce the co-immunoprecipitation signal of MCMBP and MCM3. The combination of active ingredients can bind to the MCMBP protein and reduce the interaction level between MCMBP and MCM3 proteins.

[0016] Preferably, in the in vitro cell experiments, the final concentration of each compound is 50-100 μM.

[0017] Preferably, the in vitro cell experiments use liver cancer cells. The liver cancer cells are HepG2 cells.

[0018] Preferably, the concentration of bufotin in the in vitro cell experiment is 20-400 μg / mL.

[0019] A research kit for studying the regulation of MCM3 protein expression in in vitro cell experiments, the kit comprising: bufotalin or a combination of its active ingredients, an anti-MCM3 antibody, and an anti-MCMBP antibody and / or an anti-ubiquitin antibody. The kit is used to detect the MCM3 protein expression level, MCM3 protein ubiquitination level, or MCMBP-MCM3 protein interaction level in cells treated with bufotalin or the combination of its active ingredients.

[0020] Preferably, the kit also includes cell lysis buffer, Protein A / G magnetic beads for immunoprecipitation, and Western blot assay reagent.

[0021] The beneficial effects of this invention are:

[0022] Experiments have demonstrated that bufalin can significantly inhibit the viability of tumor cells such as liver cancer cells, but has no significant effect on the viability of normal liver cells. This indicates that bufalin has a selective inhibitory effect on rapidly proliferating cells (such as tumor cells), rather than indiscriminately killing all cells. This characteristic makes it promising to overcome the lack of selectivity of traditional cytotoxic drugs and reduce toxic side effects on normal tissues. Furthermore, this invention reveals for the first time that bufalin exerts its effects by targeting the interaction between MCMBP and MCMs, providing a novel molecular explanation for the anti-tumor mechanism of bufalin and offering new lead compounds and research directions for the development of anti-tumor drugs targeting MCMs.

[0023] The mechanism of action of bufotalin in inhibiting MCMs proteins described in this invention further includes: bufotalin interferes with the assembly process of MCMs complexes, disrupts the stability of the MCM2-7 hexamer complex, thereby inhibiting the activity of DNA replication helicase and blocking DNA replication and proliferation of tumor cells.

[0024] The binding mode of the active ingredient to MCMBP protein described in this invention involves bufalin and resibufogenin, among other bufalin lactone compounds, interacting with the hydrophobic binding pocket of MCMBP through specific functional groups (such as C14-β-hydroxyl, C16-acetoxy, etc.) on their steroidal nucleus, forming a stable complex. Attached Figure Description

[0025] Figure 1This is a graph showing the detection results of the inhibition of MCMs protein expression in human hepatocellular carcinoma HepG2 by bufotoxin in Example 1 of the present invention; where A: HepG2 cells were treated with different doses of bufotoxin (0, 20, 50, 100, 200, 400 μg / mL) for 24 h, and the expression of MCMBP, MCM3, MCM5, and MCM7 proteins was detected by Western blot; B: HepG2 cells were treated with different doses of bufotoxin (0, 100, 200, 400 μg / mL) for 24 h, and the expression of MCM3, MCM5, and MCM7 mRNA was detected by qPCR (mean±SD, n=4); ns: no statistical difference.

[0026] Figure 2 This is a graph showing the detection results of bufotoxin inhibiting the interaction between MCMBP and MCM3 and promoting the degradation of MCM3 protein in Example 2 of the present invention; wherein, A: 293T cells were transfected with MYC-MCM3 and HA-Ub plasmids, and the effect of bufotoxin on the ubiquitination of MCM3 protein was detected by IP method; B: 293T cells were transfected with MYC-MCM3 and Flag-MCMBP plasmids, and the effect of bufotoxin on the binding of MCMBP and MCM3 protein was detected by co-IP method.

[0027] Figure 3 This is a flowchart and identification results diagram of the determination of the MCMBP-bound component in bufotoxin by surface plasmon resonance (SPR) in Example 3 of the present invention; wherein, A: SPR flowchart; B: Recovery curve of Control group sample (left); Recovery curve of MCMBP group sample (right); C: UPLC-HRMS base peak ion chromatogram of SPR eluent - negative ion mode; D: UPLC-HRMS base peak ion chromatogram of SPR eluent - positive ion mode; E: Identification results of the main components of SPR eluent.

[0028] Figure 4 This is a graph showing the detection results of the active ingredients of bufotoxin and their combination inhibiting the expression of MCM3 protein in human liver cancer cells HepG2 in Example 4 of the present invention; wherein, A: HepG2 cells were treated for 24 h with different doses of bufotoxin, bufotoxin, bufotoxin talidin, bufotoxin ligand, bufotoxin talidin, deacetylated bufotoxin, bufotoxin talidin, far-infrared bufotoxin, bufotoxin talidin, sea bufotoxin, and bufotoxin talidin respectively at 0, 50, and 100 μM; B: HepG2 cells were treated with a mixture of these 11 active ingredients for 24 h, and the expression of MCM3 protein was detected by Western blot.

[0029] Figure 5In Example 5 of this invention, molecular docking analysis was used to further verify the binding of the active ingredients bufotoxin, bufotoxin, bufotoxin taloxone, bufotoxin ligand, bufotoxin taloxone, deacetylated bufotoxin, bufotoxin taloxone, far-infrared bufotoxin, bufotoxin taloxone, sea bufotoxin, and bufotoxin taloxone with MCMBP protein, and the binding sites were determined.

[0030] Figure 6 This is a molecular docking analysis of the interaction between MCM3 protein and MCMBP protein in Example 6 of the present invention.

[0031] Figure 7 This is a graph showing the detection results of the effect of bufotoxin and its active ingredient combination on the viability of HepG2 and AML12 cells in Example 7 of the present invention. In the graph, A: HepG2 cells or AML12 cells were treated with different doses of bufotoxin (0, 20, 50, 100, 200, 400 μg / mL) for 24 h, and cell viability was detected by CCK8 assay (mean±SD, n=6); B: HepG2 cells or AML12 cells were treated with different doses of active ingredient mixture (0, 10, 20, 50, 100, 200 μM) for 24 h, and cell viability was detected by CCK8 assay (mean±SD, n=6). Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0033] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0034] Unless otherwise specified, the reagents used in the following examples can be purchased from regular biochemical reagent stores. The drugs, cells, and plasmids used in the following examples are as follows:

[0035] 1. Bufotalin capsules, purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd. (National Drug Approval Number Z20050846, Batch No. 4L01); Bufotalin (CAS No.: 465-21-4, Product No. V96097), Bufotalin Extract (CAS No.: 6691-83-4, Product No. V88725), Bufotalin (CAS No.: 1108-68-5, Product No. B20541), Bufotalin Glycol (CAS No.: 465-39-4, Product No. V96098), Bufotalin Extract (CAS No.: 471-95-4, Product No. V88425), Deacetylated Bufotalin Extract (CAS No.: 4026-95-3, Product No. B21036), Bufotalin Extract (CAS No.: 465-90-7, Product No. V88424), and Bufotalin Extract (CAS No.: The standard products, including 464-74-4 (item number V89230), bufotalin (CAS number: 465-11-2, item number B21070), bufotalin (CAS number: 470-42-8, item number V89053), and bufotalin (CAS number: 20987-24-0, item number V89643), were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] 2. The human hepatocellular carcinoma cell line HepG2, the human embryonic kidney cell line 293T, and the normal mouse hepatocyte cell line AML12 were all purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., and were identified by the company.

[0037] 3. The plasmids MYC-MCM3 (catalog number GOSE5043972), HA-Ub (catalog number GCPE5043974), and Flag-MCMBP (catalog number GOSE5043971) were purchased from Shanghai Jikai Gene Technology Co., Ltd.

[0038] The Huachansu described in this invention is an extract of dried toad skin, a traditional Chinese medicine. Its formulations include, but are not limited to, pharmaceutically acceptable dosage forms such as tablets, capsules, injections, granules, and oral liquids. As a marketed traditional Chinese medicine preparation, the safety of Huachansu has been verified through long-term clinical application.

[0039] The MCMs proteins described in this invention mainly include members of the mini chromosome maintenance protein family, such as MCM2, MCM3, MCM5, MCM6, and MCM7. Among them, MCM3, MCM5, and MCM7 are abnormally highly expressed in various malignant tumors and are closely related to tumor development and poor prognosis.

[0040] The mechanism of action of bufalin in inhibiting MCMs proteins described in this invention is as follows: Bufalin inhibits the interaction between MCMs proteins and their molecular chaperone MCMBP (MCM-binding protein), thereby disrupting the stability of the MCMs complex and promoting the degradation of MCMs proteins through the ubiquitin-proteasome pathway. Ultimately, this inhibits the initiation and elongation of DNA replication, thus exerting an anti-tumor effect. This mechanism is fundamentally different from the mechanism by which bufalin in existing technologies inhibits cancer cell proliferation or induces apoptosis by regulating signaling pathways such as PI3K / AKT.

[0041] The diseases associated with abnormal expression or activity of MCMs proteins described in this invention mainly include, but are not limited to, malignant tumors such as liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, ovarian cancer, and glioma. MCMs proteins are abnormally highly expressed in these diseases and are potential intervention targets.

[0042] The core of this invention lies in providing the application of bufotin or a combination of its active ingredients in the preparation of in vitro MCMs protein inhibitors for non-disease treatment purposes.

[0043] The MCMs proteins mainly include members of the microchromosome maintenance protein family, specifically including at least one of MCM3, MCM5, or MCM7.

[0044] The in vitro MCMs protein inhibitor is an MCMs protein degrader. Specifically, the application involves inhibiting the interaction between MCMs proteins and their molecular chaperone MCMBP proteins, thereby promoting the degradation of MCMs proteins via the ubiquitin-proteasome pathway.

[0045] The active ingredient combination of the bufotalin is a combination of bufotaloid compounds, specifically selected from 11 monomeric compounds including bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, arenobufagin, gamabufotalin, marinobufagin, and resibufagin. The combination can be in equal proportions or other predetermined proportions.

[0046] The combination of active ingredients, as a mixture of small molecule ligands targeting the MCMBP protein, competitively blocks the interaction between MCMs and MCMBP in vitro.

[0047] The in vitro MCMs protein inhibitor is a research reagent or in vitro biochemical detection kit used in cell biology research.

[0048] Another core aspect of this invention is providing a research kit for in vitro study of MCM protein function. The research kit contains bufotin or a combination of its active ingredients as an inhibitor targeting the MCMBP-MCM protein interaction.

[0049] Furthermore, the kit also includes detection reagents for in vitro detection of MCM protein expression levels, MCM protein ubiquitination levels, or MCMBP protein binding affinity. For example, it may contain antibodies that specifically recognize MCM3, MCM5, MCM7, or MCMBP, as well as related reagents for detecting ubiquitination.

[0050] Example 1: Bufotalin inhibits the expression of MCMs protein in human hepatocellular carcinoma cells HepG2.

[0051] The contents of Huachansu capsules (purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd.) were dissolved in DMSO and sonicated to aid dissolution. The supernatant was collected by centrifugation and prepared into different doses of Huachansu (0, 20, 50, 100, 200, 400 μg / mL) to treat HepG2 cells for 24 h. Western blot and qPCR were used to detect the protein expression of MCMBP, MCM3, MCM5, and MCM7, as well as the mRNA expression of MCM3, MCM5, and MCM7.

[0052] like Figure 1 Western blot and qPCR results showed that bufotoxin significantly reduced the abundance of MCM3, MCM5, and MCM7 proteins, validating its inhibitory effect on MCMs proteins at the in vitro cellular level. Figure 1 The results showed that bufotoxin had no significant effect on the mRNA transcription level of the MCMs family (such as MCM3, MCM5, and MCM7), but significantly reduced their protein abundance. This asynchronous phenomenon between transcription and protein expression strongly suggests that bufotoxin does not downregulate MCMs protein levels by inhibiting gene transcription, but rather through post-transcriptional regulatory mechanisms (such as reducing protein stability or promoting protein degradation).

[0053] Example 2: Bufotin inhibits the interaction between MCMBP and MCM3 and promotes the degradation of MCM3 protein.

[0054] MYC-MCM3 and HA-Ub plasmids were transfected into 293T cells, followed by treatment with bufotalin (400 μg / mL) for 24 h. IP assay confirmed that bufotalin significantly promoted MCM3 protein ubiquitination. Figure 2 A).

[0055] MYC-MCM3 and Flag-MCMBP plasmids were transfected into 293T cells, followed by treatment with bufotalin (400 μg / mL) for 24 h. The co-IP assay confirmed that bufotalin inhibited the binding of MCMBP to MCM3 protein. Figure 2 B).

[0056] MCMBP, as a molecular chaperone for MCMs, is crucial for maintaining the stability of MCMs complexes. The results indicate that bufotoxin can specifically interfere with the binding of MCM3 to its molecular chaperone MCMBP. Figure 2 B). Because MCMBP plays a crucial protective role in maintaining the stability of MCMs complexes (such as the MCM2-7 hexamer), the uncoupling of this protein-protein interaction (PPI) leads to conformational instability of the free MCM3 protein and exposure of ubiquitination sites, which are then recognized by the ubiquitin-proteasome system and degraded more rapidly. Figure 3 A). This novel mechanism of PPI inhibition and ubiquitination degradation unexpectedly achieves highly efficient clearance of DNA replication core proteins, providing a new intervention pathway for inhibiting abnormally proliferating cells (such as tumor cells).

[0057] Example 3: Compounds that directly bind to MCMBP exist in bufotoxin.

[0058] To identify the key active ingredient in bufalin that inhibits MCMBP-MCMs interaction, we dissolved the contents of bufalin capsules (purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd.) in DMSO, sonicated them, centrifuged them, collected the supernatant, and diluted it with buffer to prepare the SPR loading solution. Subsequently, using MCMBP as the target protein, we screened the protein using surface ionosphere resonance (SPR). Figure 2 A). The results showed that the binding process of bufotoxin to MCMBP protein exhibited a significant increase in response value, confirming the presence of a component in bufotoxin that can directly bind to MCMBP. Figure 3 B). Further UPLC-Q-TOF-MS analysis of the binding eluent identified 12 compounds as potential binding components, including bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, arenobufagin, gamabufotalin, marinobufagin, resibufaginol, and resibufagin. Figure 3(C-3E). The specific active components in bufotoxin that bind to MCMBP protein were identified.

[0059] Example 4: Bufotin monomers had no significant inhibitory effect on MCM3 protein, but their combination significantly inhibited MCM3 protein.

[0060] Monomer activity screening: HepG2 cells were treated with 0, 50, and 100 μM of bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, arenobufagin, gamabufotalin, marinobufagin, and resibufagin for 24 h, respectively. Western blot was used to detect MCM3 protein expression. The results showed that none of the 11 bufalin monomers significantly inhibited MCM3 protein expression. Figure 4 A). Since bufotalin is present in extremely low concentrations and there is currently no commercially available standard, no experimental verification was performed.

[0061] Preparation and treatment of the mixture: 2 μL of each of the 11 monomers (100 mM stock solution) were mixed to obtain a mixed stock solution (each monomer concentration in the stock solution was 9.09 mM). The mixed stock solution was diluted with culture medium and used to treat HepG2 cells for 24 h. The concentration of the mixture was calculated as the cumulative total concentration of the 11 components. The low-dose group had a total concentration of 50 μM (corresponding to a final concentration of 4.545 μM for each monomer), and the high-dose group had a total concentration of 100 μM (corresponding to a final concentration of 9.09 μM for each monomer). Western blot analysis was used to detect MCM3 protein expression. The results showed that the mixture significantly inhibited MCM3 protein expression. Figure 4 B).

[0062] The above results indicate that, even at concentrations far below the effective exploratory concentrations (50 and 100 μM) for each monomer alone (only 4.5–9 μM in the composition), the composition consisting of 11 components can significantly inhibit MCM3 protein. This demonstrates that the inhibition of MCM3 by bufalin does not depend on a single highly active ingredient, but rather stems from unexpected synergistic functional complementarity among multiple components, revealing the unique material basis for the "multi-component synergistic" anti-tumor effect of bufalin as a traditional Chinese medicine.

[0063] Example 5: Molecular docking analysis of 11 active components of Huachansu with MCMBP protein

[0064] The three-dimensional structures of bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, arenobufagin, gamabufotalin, marinobufagin, and resibufagin were obtained from the PubChem database in SDF format and converted to *mol2* format using Open Babel. The predicted three-dimensional structure of MCMBP (AF-Q9BTE3-3-F1-v6) was downloaded from the AlphaFold database. Subsequently, the ligand and protein structures were converted to *pdbqt* format using AutoDock Tools. Molecular docking simulations were performed using AutoDock Vina with default parameters. Docking conformation and protein-ligand interactions were analyzed and visualized using PyMOL.

[0065] The results showed that the binding energies of bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, arenobufagin, gamabufotalin, marinobufagin, and resibufagin to MCMBP protein were -8, -8.4, -8.5, -8.5, -8.3, -8.2, -8.4, -8.3, -8.6, -8.5, and -8.6 kcal / mol, respectively, indicating strong binding. The binding diagrams of each monomeric compound to MCMBP protein are shown below. Figure 5 As shown. Furthermore, molecular docking results indicate that the interaction sites between these 11 monomeric compounds and the MCMBP protein are mainly on amino acid residues such as TYR, GLY, ARG, LYS, and GLU. Figure 5 ).

[0066] Example 6: Interaction analysis between MCM3 and MCMBP proteins

[0067] Download the predicted 3D structure of MCM3 (PDB ID: 22VT) from the PDB database and MCMBP (AF-Q9BTE3-3-F1-v6) from the AlphaFold database. (via HDOCK website) http: / / hdock.phys.hust.edu.cn / Protein-protein docking simulations were performed to analyze different docking conformations, binding activity in different conformations, and amino acid residues with interaction distances within 5 Å. The three-dimensional structures of the proteins were preprocessed using Pymol software to plot the amino acid residues involved in the interaction between the two proteins. The top 10 conformations were sorted by HDOCK docking score for analysis. Figure 6 A). The results showed that the main amino acid residues interacting between MCM3 and MCMBP proteins were TYR, GLY, ARG, LYS, and GLU. Figure 6 B).

[0068] comprehensive Figure 5 and Figure 6 The results showed that all 11 monomeric compounds and MCM3 contacted certain identical amino acid residues on the MCMBP. This high degree of consistency in the types of residues involved indicates that these 11 monomeric components can collectively target key residue regions on the MCMBP closely related to MCM3 binding; when they act together, they can effectively block the binding of MCM3 to MCMBP by synergistically occupying or perturbing these co-dependent amino acid residues, creating multiple spatial and energy interferences. This fully confirms that the inhibitory effect of bufotalin on MCM3-MCMBP binding is the result of the synergistic effect of these 11 components.

[0069] Example 7: Huachansu significantly inhibited the activity of liver cancer cells, but had no effect on the activity of normal liver cells.

[0070] Human hepatocellular carcinoma HepG2 cell lines or normal mouse AML12 hepatocytes were treated for 24 h with different doses of bufotalin (0, 20, 50, 100, 200 μg / mL) or mixtures of active ingredients (0, 10, 20, 50, 100, 200 μM), and cell viability was detected by the CCK8 assay. The preparation method of the mixtures of active ingredients was the same as in Example 4, and the concentration of the mixtures was expressed as the cumulative total concentration of the 11 components.

[0071] Figure 7 The CCK8 assay results showed that bufotalin and its active ingredient combination reduced HepG2 cell viability in a dose-dependent manner, but had no effect on AML12 hepatocyte viability. This indicates that bufotalin and its active ingredient combination have a highly selective inhibitory effect on rapidly proliferating cells, thereby reducing toxicity to normal cells while ensuring efficacy.

[0072] This remarkable cell selectivity aligns perfectly with the biological characteristics of MCMs proteins: malignant tumor cells (such as HepG2) experience extremely high DNA replication pressure and are highly dependent on MCMs helicase complexes to maintain proliferation; while normal hepatocytes (such as AML12) are mostly in a quiescent phase (G0 phase) and have lower dependence on MCMs. Therefore, Huachansu, by targeting and degrading MCMs proteins, can precisely inhibit tumor cell replication while exhibiting good safety for normal cells.

[0073] In summary, this invention is the first to discover that bufotoxin and its combination of active ingredients can act as direct inhibitors of MCMs proteins, promoting the ubiquitination and degradation of MCMs proteins by targeting the MCMBP-MCMs protein interaction. This discovery not only reveals a novel molecular mechanism of bufotoxin's anti-tumor effect but also provides new lead compounds and theoretical basis for the development of anti-tumor drugs targeting MCMs, while providing a powerful research tool for cell biology research.

Claims

1. The use of a bufotin or a combination of its active ingredients in the preparation of an in vitro MCM protein inhibitor for non-disease treatment purposes.

2. The application according to claim 1, characterized in that, The MCMs proteins include at least one of MCM3, MCM5, or MCM7.

3. The application according to claim 1, characterized in that, The in vitro MCMs protein inhibitor is an MCMs protein degrader; the specific application is: by inhibiting the interaction between MCMs protein and its molecular chaperone MCMBP protein, the degradation of MCMs protein is promoted through the ubiquitin-proteasome pathway.

4. The application according to claim 1, characterized in that, The active ingredient combination of the bufotalin is a bufotalin-based compound, consisting of 11 monomeric compounds including bufalin, cinobufagin, cinobufotalin, resibufogenin, bufotalin, deacetylcinobufagin, bufotalidin, areenobufagin, gamabufotalin, marinobufagin, and resibufagin.

5. The application according to claim 4, characterized in that, The active ingredient combination is a combination of small molecule ligands that target the MCMBP protein, competitively blocking the interaction between MCMs and MCMBP in vitro.

6. The application according to claim 1, characterized in that, The in vitro MCMs protein inhibitor is a research reagent or in vitro biochemical detection kit used in cell biology research.

7. The application of a bufotoxin or a combination of its active ingredients in the preparation of a research reagent for reducing the expression level of MCM3 protein in in vitro cell experiments for non-disease treatment purposes, wherein the combination of active ingredients comprises bufotoxin, bufotoxin, bufotoxin talidin, bufotoxin ligand, bufotoxin talidin, deacetylated bufotoxin, bufotoxin talidin, bufotoxin talidin, bufotoxin talidin, bufotoxin talidin, and bufotoxin talidin.

8. The application according to claim 7, characterized in that, The research reagents are also used to reduce the expression levels of MCM5 and / or MCM7 proteins.

9. A research kit for studying the regulation of MCM3 protein expression in in vitro cell experiments, characterized in that, The kit includes: bufotoxin or the combination of active ingredients as described in claim 4, anti-MCM3 antibody, and anti-MCMBP antibody and / or anti-ubiquitin antibody.

10. The research kit according to claim 9, characterized in that, The kit also includes cell lysis buffer, Protein A / G magnetic beads for immunoprecipitation, and Western blot detection reagent.

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