Use of a method for inhibiting proliferation of hcc cells as a potential inhibitor molecule of pcid2

Abstract

The present application relates to a kind of inhibitor molecules with PCID2 as action target and its application, belong to tumor related molecular regulation and biological medicine technical field.The candidate small molecule with multiple hydroxyl substituent group and rigid cyclic skeleton structure is constructed, so that it can occur stable interaction with PCID2 protein key binding site, to reduce the functional activity of PCID2, to regulate the cell proliferation related signal pathway mediated by it.The inhibitor molecule can be screened and verified by molecular docking etc., and can be prepared into corresponding drug or biological active composition.Research shows that the inhibitor molecule exhibits the biological effect of inhibiting abnormal cell proliferation in PCID2 high expression related model, while having good biological safety and acceptable in vivo metabolic characteristics, which provides a new technical scheme and application basis for the intervention of PCID2 related diseases.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of biomedical technology, and particularly relates to the use of a method for inhibiting HCC cell proliferation as a potential PCID2 inhibitor molecule. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, characterized by insidious onset, high malignancy, rapid progression, and poor prognosis. Its mortality rate is close to its incidence rate, with a 5-year survival rate of only 18%. Current treatments for HCC primarily include liver resection, liver transplantation, local therapies (including ablation, transarterial chemoembolization, and radiotherapy), immunotherapy, nanotherapy, and systemic therapy. The specific treatment plan is determined by the clinical stage. Due to the insidious onset, rapid progression, and high malignancy of early-stage HCC, most HCC patients (approximately 50%) are diagnosed at an intermediate or even terminal stage, making them ineligible for surgical treatment and resulting in a very poor prognosis. Therefore, the treatment of HCC has limitations.

[0003] Systemic antitumor therapy plays a crucial role in the treatment of intermediate-to-advanced hepatocellular carcinoma (HCC). It is the primary, and sometimes only, treatment option for patients with non-surgically resected HCC, controlling disease progression, prolonging survival, and achieving partial or complete remission in some patients. Although the advent of molecularly targeted drugs represents a major breakthrough in HCC treatment, and the prospects for advanced hepatocellular carcinoma are more diverse than ever before, the overall prognosis for HCC patients remains poor, and survival rates have not significantly improved. Furthermore, the safety and resistance of these drugs require attention during treatment and necessitate further research.

[0004] Therefore, research on the core regulatory factors and specific target drugs of HCC is of great significance for the treatment of liver cancer and the improvement of prognosis. The development of natural small molecule compounds is a promising direction for molecular targeted therapy of tumors. Targeting specific factors that regulate the cell cycle can provide new concepts and directions for targeted tumor therapy and is also key to improving tumor prognosis. Finding specific targets that regulate the tumor cell cycle and discovering related lead compounds are promising strategies for combating liver cancer.

[0005] Recent studies have shown that PCI domain-containing protein 2 (PCID2) is a key protein regulating cell cycle checkpoints and has biological functions in maintaining the stemness of embryonic stem cells and regulating their self-renewal and differentiation. It is a key factor in regulating the development and progression of liver cancer and a potential target for anti-HCC drugs. Developing inhibitor molecules targeting PCID2 has important research value and can provide new strategies for HCC treatment.

[0006] However, research on inhibitors targeting PCID2 is still in its early stages, and no inhibitor molecules specifically targeting PCID2 have been publicly reported. This may be related to the fact that the function of PCID2 has not been fully elucidated and the lack of efficient technical means to screen inhibitors.

[0007] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0008] (1) Limited treatment options: Treatment of HCC usually relies on traditional methods such as surgery, radiotherapy and chemotherapy, which are often accompanied by serious side effects and have limited effectiveness for patients in advanced stages.

[0009] (2) Individual differences in drug efficacy: Due to differences in patients’ genetic background and tumor biomarkers, the efficacy of existing drug treatments often varies significantly among individuals.

[0010] (3) Drug resistance problem: HCC patients often develop drug resistance after receiving chemotherapy, leading to treatment failure.

[0011] (4) Lack of targeted drugs: Although some targeted drugs have been developed for the treatment of HCC, there is still a lack of highly effective drugs targeting specific molecular targets. Summary of the Invention

[0012] In view of the problems existing in the prior art, the present invention provides a method for inhibiting HCC cell proliferation as a potential PCID2 inhibitor molecule.

[0013] This invention provides a PCID2 inhibitor molecule that can specifically bind to the functional binding site of the PCID2 protein, thereby reducing the functional activity of the PCID2 protein.

[0014] The inhibitor molecule comprises multiple hydroxyl substituents and a rigid cyclic backbone structure, wherein:

[0015] Polyhydroxy substituents are used to form a stable hydrogen bond network with key amino acid residues of PCID2;

[0016] The rigid ring-shaped framework is used to form a spatially complementary hydrophobic interaction with the PCID2 hydrophobic pocket.

[0017] Furthermore, the inhibitor molecule can significantly reduce the functional activity or effective expression level of PCID2 protein in cells.

[0018] Furthermore, the inhibitor molecules inhibit abnormal cell proliferation behavior by interfering with PCID2-mediated cell proliferation regulation-related signaling pathways.

[0019] This invention achieves an innovative drug application for HCC (hepatocellular carcinoma) by using Theaflavin 3,3'-digallate (TF-3) to inhibit HCC cell proliferation. TF-3 exerts a regulatory effect on HCC cell proliferation, migration, invasion, and apoptosis through a unique molecular mechanism of action that inhibits PCID2 expression, thereby exhibiting significant anti-hepatocellular carcinoma activity. This invention provides a potential novel therapeutic approach, opening new directions for HCC intervention and treatment. TF-3 can significantly downregulate the protein expression levels of cell cycle-related proteins (Cyclin D1 and CDK6) and significantly reduce the protein expression of key core factors in the PI3K / Akt / NF-κB signaling pathway by inhibiting PCID2 expression. This mechanism of action achieves precise regulation of HCC cells, providing a scientific basis for its treatment of HCC.

[0020] This invention is achieved as follows: a method for inhibiting HCC cell proliferation as a potential PCID2 inhibitor molecule includes:

[0021] Step 1: By applying candidate small molecules with PCID2 inhibitory activity to HCC cells, the expression level or functional activity of PCID2 protein is reduced.

[0022] Step 2 leads to the inhibition of cell proliferation regulatory pathways related to PCID2, thereby reducing the proliferative capacity of hepatocellular carcinoma cells at the cellular level.

[0023] Furthermore, the hepatocellular carcinoma cells are in vitro cultured cells derived from human hepatocellular carcinoma.

[0024] Furthermore, the cell proliferation inhibition effect was verified using cell viability detection methods and cell colony formation ability detection methods;

[0025] The target specificity of the inhibitory effect was confirmed by detecting changes in PCID2 protein expression levels or functional activity.

[0026] Furthermore, the method for constructing the candidate small molecule is as follows:

[0027] The method uses PCID2 as a functional inhibitory target and constructs candidate small molecules that can inhibit the biological function of PCID2 by leveraging the synergistic matching relationship between structural features and target binding sites.

[0028] The collaborative matching relationship includes:

[0029] In the candidate molecule, multiple hydroxyl groups form a stable hydrogen bond network with key amino acid residues of PCID2;

[0030] The cyclic backbone in the candidate molecule forms a spatially complementary hydrophobic interaction with the PCID2 hydrophobic pocket;

[0031] This allows for the inhibition of PCID2 activity at the molecular level, and through this inhibition, the proliferation signaling pathway of hepatocellular carcinoma cells is blocked.

[0032] Furthermore, the candidate small molecules are obtained by screening from existing compound libraries, and the screening criteria are that the candidate molecules simultaneously possess a polyhydroxy substitution structure and a rigid cyclic skeleton.

[0033] Furthermore, the cooperative matching relationship is verified through molecular docking calculations;

[0034] The molecular docking calculations are used to determine the binding conformational stability and key interaction sites between candidate molecules and PCID2.

[0035] Another objective of this invention is to provide a method for evaluating the safety and in vivo behavior of PCID2 inhibitor molecules, after confirming that the candidate molecule possesses PCID2 inhibitory activity;

[0036] The biosafety and in vivo action characteristics of candidate molecules were evaluated synergistically through systematic toxicological assessment and in vivo metabolic behavior analysis.

[0037] To determine the feasibility of the candidate molecule in inhibiting the proliferation of hepatocellular carcinoma cells.

[0038] Furthermore, the toxicological assessment includes acute toxicity evaluation and chronic toxicity evaluation of candidate molecules to determine their biosafety range.

[0039] Furthermore, the in vivo metabolic behavior analysis includes a systematic study of the absorption characteristics, in vivo distribution characteristics, metabolic characteristics, and excretion characteristics of candidate molecules.

[0040] Furthermore, by monitoring tumor growth changes in an animal model of hepatocellular carcinoma, the in vivo antitumor effects of candidate molecules were correlated with their safety evaluation results to verify their comprehensive application value.

[0041] Another objective of this invention is to provide a method for discovering TF-3's inhibition of PCID2 using a virtual screening technique based on molecular docking, and for evaluating the binding affinity of TF-3 and PCID2 in vitro, comprising the following steps:

[0042] (1) Based on the crystal structure of Human PCID2 (PDB code: 3T5X) in the Protein Data Bank database, the active site was determined, the grid parameters were set, and the binding mode of the known inhibitor molecule FO (N-trans-feruloyloctopamine) to PCID2 was verified.

[0043] (2) Using molecular docking technology, the TF-3 molecular structure was docked to the PCID2 active site. The docking score and binding mode were analyzed and compared with the binding mode of the known inhibitor molecule FO to determine its binding potential.

[0044] (3) The binding ability of TF-3 and PCID2 was evaluated in vitro using surface plasmon resonance (SPR); the interaction between TF-3 and PCID2 in the intracellular environment was verified using cell thermal shift aastomosis (CETSA).

[0045] Another object of the present invention is to provide a cell verification method for TF-3 to exert anti-HCC activity in vitro, comprising the following steps:

[0046] (1) The effects of TF-3 intervention on the proliferation of HepG2 and Huh7 cells were detected by CCK-8 assay and EdU assay respectively.

[0047] (2) The colony formation assay was used to further evaluate the inhibitory effect of TF-3 on the long-term proliferation of HepG2 and Huh7 liver cancer cells.

[0048] (3) The effect of TF-3 intervention on the invasive ability of HepG2 and Huh7 liver cancer cells was detected by Transwell cell invasion assay.

[0049] (4) The effect of TF-3 intervention on the migration ability of HepG2 and Huh7 liver cancer cells was evaluated by cell scratch assay.

[0050] (5) The Annexin V-FITC / PI double staining method was used to detect the effect of TF-3 intervention on apoptosis of HepG2 and Huh7 liver cancer cells.

[0051] (6) The effect of TF-3 intervention on the cell cycle of HepG2 and Huh7 liver cancer cells was detected by flow cytometry.

[0052] Another object of the present invention is to provide an experimental method for evaluating the safety of TF-3 at in vitro / in vivo levels, comprising the following steps:

[0053] (1) The effect of TF-3 intervention on cell viability was detected in normal human hepatocytes using the CCK-8 assay;

[0054] (2) A mouse model of subcutaneous xenograft liver cancer was constructed using male BABL / c mice, and the mice were randomly divided into: blank control group, model group, and low, medium and high dose TF-3 groups;

[0055] (3) After establishing a subcutaneous xenograft hepatocellular carcinoma mouse model, the mice were fed normally for 2 weeks before starting drug administration. The control group received intraperitoneal injection of the solvent; the intervention group was administered the drug based on the IC50 obtained from cell experiments. 50 The values ​​were converted to appropriate intervention doses for mice, diluted to suitable concentrations, and administered 100 μL intraperitoneally once daily for two weeks at low (5 mg / kg / day), medium (10 mg / kg / day), and high (20 mg / kg / day) levels. The control group and model group were fed normally.

[0056] (4) Observe the mice’s condition (such as hair and mental state) throughout the experiment, and measure and record the changes in the weight of mice in each group.

[0057] (5) Two weeks after TF-3 intervention, blood was collected from the eyeballs, centrifuged to obtain serum, and the levels of liver-related serum metabolites such as alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), α-L-fucosidase (AFU) and total bile acid (TBA) were detected using an AU5800 fully automated biochemical analyzer.

[0058] (6) After the mice were sacrificed, the in situ tumor block in the liver, liver, lungs and kidneys were removed and the tumor block and tissues of each organ were observed with the naked eye to evaluate the effect of TF-3 intervention on the organs and tissues of mice.

[0059] Another objective of this invention is to provide a cell experimental method for elucidating the mechanism by which TF-3 inhibits PCID2 and blocks the proliferation of liver cancer cells, comprising the following steps:

[0060] (1) The changes in PCID2 protein expression levels after TF-3 intervention in two hepatocellular carcinoma cell lines, HepG2 and Huh7, were detected by Western Blot.

[0061] (2) In HepG2 and Huh7 hepatocellular carcinoma cell lines, the changes in the expression levels of cell cycle proteins (Cyclin D1 and CDK6) after TF-3 intervention in the two hepatocellular carcinoma cell lines were detected by Western Blot.

[0062] (3) In HepG2 and Huh7 hepatocellular carcinoma cell lines, the changes in protein expression levels of core factors of the PI3K / Akt / NF-κB signaling pathway after TF-3 intervention were detected by Western Blot.

[0063] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0064] This invention discovers that Theaflavin 3,3'-digallate (TF-3) can target PCID2 and can be used to prepare PCID2 inhibitors. Furthermore, TF-3 can significantly inhibit the proliferation, migration, and invasion of HepG2 and Huh7 liver cancer cells, and promote apoptosis of liver cancer cells, exhibiting significant anti-HCC cell activity. TF-3 achieves its anti-HCC cell activity by regulating the protein expression levels of core factors in the PI3K / Akt / NF-κB signaling pathway through this unique molecular mechanism of inhibiting PCID2 expression. Moreover, TF-3 has not been found to be toxic and has good safety, making it suitable for preparing drugs to treat HCC. This invention provides a potential novel therapeutic approach, opening up new directions for the intervention and treatment of HCC.

[0065] First, PCID2 is a protein that plays a key role in regulating HCC cell proliferation; therefore, inhibitors targeting this protein are expected to provide effective treatment. This invention focuses on PCID2, a novel drug target, and demonstrates the feasibility of inhibiting PCID2 to block HCC cell proliferation, providing a novel treatment strategy for HCC.

[0066] Secondly, this invention uses TF-3 as the research object and, based on molecular docking technology combined with experimental techniques, verifies the mechanism by which TF-3 inhibits HCC cell proliferation by suppressing PCID2, providing a potential new treatment approach and offering new ideas for the intervention and treatment of HCC.

[0067] Third, this invention provides the application of TF-3 or a pharmaceutically acceptable salt thereof in the preparation of PCID2 target inhibitors, wherein the structural formula of TF-3 is as follows: Figure 2 As shown:

[0068] Preferably, the TF-3 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable excipient or carrier to form any pharmaceutically acceptable dosage form.

[0069] Preferably, the dosage form includes tablets, injections, and capsules.

[0070] Fourth, this invention provides the application of TF-3 or a pharmaceutically acceptable salt thereof in the preparation of drugs that precisely target HCC cells, thereby reducing damage to normal cells and lowering treatment-related side effects. The structural formula of the TF-3 is as follows: Figure 2 As shown:

[0071] Preferably, the TF-3 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable excipient or carrier to form any pharmaceutically acceptable dosage form.

[0072] Preferably, the dosage form includes tablets, injections, and capsules.

[0073] Fifth, this invention has discovered through molecular docking technology that TF-3 can bind well in the active pocket of PCID2 and has a similar binding mode to the PCID2 inhibitor molecule FO, proving that TF-3 has the potential to inhibit PCID2.

[0074] Sixth, this invention uses SPR and CETSA experiments to verify at the in vitro cellular level that TF-3 and PCID2 have a strong binding affinity.

[0075] Seventh, this invention has confirmed that TF-3 can effectively inhibit the proliferation, migration and invasion of liver cancer cells and promote apoptosis of liver cancer cells by different liver cancer cell lines HepG2 and Huh7, thus proving that TF-3 has anti-HCC activity.

[0076] Eighth, this invention used Western blotting to detect a significant decrease in the expression level of PCID2 protein, a significant decrease in the expression levels of cyclin D1 and CDK6, and a significant decrease in the expression levels of p-PI3K, p-AKT, and p-NF-κB proteins in HepG2 and Huh7 liver cancer cell lines after TF-3 intervention. This demonstrates that TF-3 can inhibit the proliferation of liver cancer cells by suppressing PCID2.

[0077] Ninth, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0078] (1) Expected benefits and commercial value of the technical solution of the present invention after transformation

[0079] With the deepening of scientific research and the verification of practical experience, people's attitudes towards cancer have gradually shifted from "fear of cancer" to "coexisting with cancer." However, even now, cancer remains a major public health problem worldwide and a leading contributor to the global disease burden. Primary liver cancer is one of the most serious malignant diseases posing a severe threat to global human health. According to the latest cancer data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2022, primary liver cancer ranked third among new cancer cases and cancer deaths globally (approximately 760,000 cases, accounting for 7.8%), and nearly half (49.2%) of new cancer cases and more than half (56.1%) of cancer deaths worldwide in 2022 occurred in Asia. The cancer situation in China is also not optimistic. In December 2023, a research team from the Chinese Center for Disease Control and Prevention, based on data from over 300 million Chinese people, summarized the national cancer burden trend from 2005 to 2020. They found that the total number of cancer-related deaths increased by 21.6%, with liver cancer ranking fourth in incidence among malignant tumors and second in mortality. According to data released by the National Cancer Center of China, in 2022, there were 367,700 new cases of primary liver cancer in China, ranking fourth among new malignant tumor cases. The number of deaths (316,500) and the mortality rate both ranked second, seriously threatening the health and lives of the Chinese people.

[0080] The TF-3 described in this invention is a phenolic compound extracted from black tea and is one of the main components of theaflavins. It is produced through further catalysis after tea fermentation generates catechins. Theaflavins are natural tea pigments with multiple effects, including anti-inflammatory, antiviral, antitumor, and cardiovascular improvement. Literature review shows that TF-3, as a major component of theaflavins, has made significant discoveries in preventing osteoporosis, alleviating lipid accumulation in hepatocytes, lowering blood sugar, and delaying ovarian aging; however, no pharmacological effects of TF-3 against liver cancer have been found. Therefore, the TF-3 described in this invention may have greater innovation and broader application prospects in inhibiting liver cancer, providing a new direction for research on the anti-HCC effects of natural compounds.

[0081] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally:

[0082] This invention provides a novel inhibitor targeting PCID2, offering an innovative approach to the treatment of hepatocellular carcinoma. This approach not only potentially improves treatment efficacy but also reduces side effects and addresses drug resistance issues associated with existing therapies, bringing hope to liver cancer patients. These technological advancements have significant industrial application value in drug development, paving new paths for the improvement and development of future liver cancer treatments. Attached Figure Description

[0083] Figure 1 This is a flowchart of a method for inhibiting HCC cell proliferation as a potential PCID2 inhibitor molecule provided in an embodiment of the present invention.

[0084] Figure 2 This is a molecular structure diagram of compound TF-3 provided in an embodiment of the present invention.

[0085] Figure 3 This is a schematic diagram of the molecular docking results of TF-3 and PCID2 crystal structures provided in an embodiment of the present invention.

[0086] Figure 4 These are the results of the determination of the interaction and binding kinetics between TF-3 and PCID2 provided in the embodiments of the present invention.

[0087] Figure 5 The cell survival rate of HepG2 (A) and Huh7 (B) liver cancer cells provided in this embodiment of the invention is ( ). ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0088] Figure 6 The survival rate and proliferation of HepG2 and Huh7 cells treated with TF-3 as provided in this embodiment of the invention were detected by EdU. ±S, n=3; scale bar: 500 µm). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0089] Figure 7 The clonogenic assay provided in this embodiment of the invention detects the effect of TF-3 intervention on the clonogenic ability of HepG2 and Huh7 liver cancer cells, respectively. ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0090] Figure 8 The Transwell cell invasion assay provided in this embodiment of the invention was used to detect the effect of TF-3 intervention on the invasive ability of HepG2 and Huh7 liver cancer cells, respectively. ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0091] Figure 9 The cell scratch assay provided in this embodiment of the invention is used to detect the effect of TF-3 intervention on the migration ability of HepG2 and Huh7 liver cancer cells, respectively. ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0092] Figure 10 The present invention provides flow cytometry analysis to detect the effect of TF-3 intervention on apoptosis in HepG2 and Huh7 liver cancer cells. ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0093] Figure 11 The present invention provides flow cytometry analysis of the effects of TF-3 intervention on the cell cycle of HepG2 and Huh7 liver cancer cells. ±S, n=3). *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0094] Figure 12 This invention provides an embodiment of the Western blotting method to detect the expression level of PCID2 protein after TF-3 intervention in HepG2 and Huh7 liver cancer cells. *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0095] Figure 13 This invention provides an embodiment of the Western blotting method for detecting the expression levels of the cell cycle-related protein Cyclin D1 / CDK6 after TF-3 intervention in HepG2 and Huh7 liver cancer cells. *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0096] Figure 14 This invention provides an embodiment of the Western blotting method to detect the protein expression levels of key factors in the PI3K / AKT / NF-κB signaling pathway after TF-3 intervention in HepG2 and Huh7 liver cancer cells. *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0097] Figure 15 This invention provides an embodiment of the CCK-8 assay to detect the effect of TF-3 on the proliferation of normal human hepatocytes. *P<0.05, **P<0.01 indicates a statistically significant difference compared to the control group.

[0098] Figure 16 The examples provided in this invention are the subcutaneous xenograft liver cancer mouse model (A) and the changes in body weight of the mouse model and the drug-treated group after TF-3 intervention (B).

[0099] Figure 17The images provided in this embodiment of the invention are (A) schematic diagrams of mouse liver, kidney and lung tissue after TF-3 intervention (n=6) and (B) HE staining results of mouse liver and kidney.

[0100] Figure 18 The changes in serum biochemical levels in mice treated with TF-3 according to the embodiments of the present invention are shown in the figure (n=6). *P<0.05, **P<0.01 indicates that the difference is statistically significant compared with the model group. Detailed Implementation

[0101] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0102] like Figure 1 As shown in the embodiment of the present invention, a method for inhibiting HCC cell proliferation as a potential PCID2 inhibitor molecule is provided:

[0103] S101 reduces the expression level or functional activity of PCID2 protein by acting on candidate small molecules with PCID2 inhibitory activity on hepatocellular carcinoma cells.

[0104] S102, which leads to the inhibition of cell proliferation regulatory pathways related to PCID2; thereby reducing the proliferative capacity of hepatocellular carcinoma cells at the cellular level.

[0105] The hepatocellular carcinoma cells provided in this embodiment of the invention are in vitro cultured cells derived from human hepatocellular carcinoma.

[0106] The cell proliferation inhibition effect is verified by the cell viability detection method and the cell colony formation ability detection method provided in this embodiment of the invention;

[0107] The target specificity of the inhibitory effect was confirmed by detecting changes in PCID2 protein expression levels or functional activity.

[0108] The method for constructing candidate small molecules provided in this embodiment of the invention:

[0109] The method uses PCID2 as a functional inhibitory target and constructs candidate small molecules that can inhibit the biological function of PCID2 by leveraging the synergistic matching relationship between structural features and target binding sites.

[0110] The collaborative matching relationship includes:

[0111] In the candidate molecule, multiple hydroxyl groups form a stable hydrogen bond network with key amino acid residues of PCID2;

[0112] The cyclic backbone in the candidate molecule forms a spatially complementary hydrophobic interaction with the PCID2 hydrophobic pocket;

[0113] This allows for the inhibition of PCID2 activity at the molecular level, and through this inhibition, the proliferation signaling pathway of hepatocellular carcinoma cells is blocked.

[0114] The candidate small molecules provided in this embodiment of the invention are obtained by screening from existing compound libraries. The screening criteria are that the candidate molecules simultaneously possess a polyhydroxy substitution structure and a rigid cyclic skeleton.

[0115] The collaborative matching relationship provided in this embodiment of the invention is verified by molecular docking calculation.

[0116] The molecular docking calculations are used to determine the binding conformational stability and key interaction sites between candidate molecules and PCID2.

[0117] This invention provides a method for evaluating the safety and in vivo behavior of PCID2 inhibitor molecules, after confirming that the candidate molecule has PCID2 inhibitory activity;

[0118] The biosafety and in vivo action characteristics of candidate molecules were evaluated synergistically through systematic toxicological assessment and in vivo metabolic behavior analysis.

[0119] To determine the feasibility of the candidate molecule in inhibiting the proliferation of hepatocellular carcinoma cells.

[0120] The toxicological assessment provided in this embodiment of the invention includes acute toxicity evaluation and chronic toxicity evaluation of candidate molecules to determine their biosafety range.

[0121] The in vivo metabolic behavior analysis provided in this invention includes a systematic study of the absorption characteristics, in vivo distribution characteristics, metabolic characteristics, and excretion characteristics of candidate molecules.

[0122] The embodiments of this invention provide a method to monitor tumor growth changes in an animal model of hepatocellular carcinoma and then perform correlation analysis between the in vivo antitumor effects of candidate molecules and their safety evaluation results to verify their comprehensive application value.

[0123] This invention provides a novel inhibitor targeting PCID2, offering an innovative approach to the treatment of hepatocellular carcinoma. This approach not only potentially improves treatment efficacy but also reduces side effects and addresses drug resistance issues associated with existing therapies, bringing hope to liver cancer patients. These technological advancements have significant industrial application value in drug development, paving new paths for the improvement and development of future liver cancer treatments.

[0124] Experiment of this invention:

[0125] Experiment 1

[0126] 1. Experimental Instruments and Materials

[0127] 1.1 Cells and animals used in the experiment

[0128] Human HepG2 liver cancer cell line was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. BALB / c nude mice (SPF grade) used in the experiments were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., certificate number: SCXK(Su)2020-0009, experimental animal use license number: SYXK(Gan)2018-0002. The experimental mice (5-6 weeks old, weighing 22±2 g) were housed in the SPF-grade animal room of the Medical Experimental Center of the School of Basic Medical Sciences, Lanzhou University, under 12-day alternating light, with bedding, feed, and drinking water changed every 2-3 days. All animals had free access to food and water. The management, care, and ethical welfare of the experimental animals were carried out in accordance with the "Guiding Opinions on the Humane Treatment of Experimental Animals" issued by the Ministry of Science and Technology of China and the national standard "Guidelines for Ethical Review of Experimental Animal Welfare" (GB / T35892-2018). The animal experimental procedures complied with the regulations of the Ethics Committee of the First Hospital of Lanzhou University, ethics approval number LDYYLL2021-78.

[0129] 1.2 Experimental Apparatus

[0130] Table 1 Experimental Apparatus

[0131]

[0132] 1.3 Experimental Reagents

[0133] Table 2 Experimental Reagents

[0134]

[0135] 1.4 Experimental Methods

[0136] 1.4.1 Construction of a mouse model of subcutaneous xenograft hepatocellular carcinoma

[0137] Cell preparation: Collect HepG2 cells in logarithmic growth phase, digest normally, count, wash twice with PBS, and finally resuspend in PBS to adjust the cell concentration to 1×10⁻⁶. 7 Quantity / mL, ready for use.

[0138] Establishment of a mouse model of subcutaneous xenograft hepatocellular carcinoma: Mice were anesthetized by intraperitoneal injection of 0.1 mL of 10% chloral hydrate and fixed in a supine position. Each mouse was subcutaneously injected with 0.2 mL of a solution containing 1×10⁻⁶ chloral hydrate under the armpit. 7 A suspension of individual cells was prepared. Mice survival and tumor growth were monitored and recorded weekly. After two weeks, mice were euthanized, tumor tissue was removed, and photographs and weighing were performed.

[0139] 1.4.2 Animal grouping and administration methods

[0140] BABL / c nude mice were randomly divided into four groups: a blank control group, a model group, a low-dose TF-3 group (L), a medium-dose TF-3 group (M), and a high-dose TF-3 group (H), with six mice in each group. After establishing a subcutaneous xenograft hepatocellular carcinoma mouse model, mice were fed normally for two weeks before starting drug administration. The control group received intraperitoneal injection of the solvent; the intervention group was evaluated based on the IC50 obtained from cell experiments. 50 The values ​​were converted to appropriate intervention doses for mice, diluted to suitable concentrations, and administered 100 μL intraperitoneally once daily for 2 weeks at low (5 mg / kg / day), medium (10 mg / kg / day), and high (20 mg / kg / day) concentrations. The control group and model group were fed normally and observed at appropriate times.

[0141] After the animal model was established, the mouse weight, hair and mental state were measured on the 7th day after inoculation. The measurements were then taken every 2-3 days and recorded each time.

[0142] 1.4.3 Collection of serum and tissue samples

[0143] Two weeks after intervention with the test compound, the mouse head was fixed, the skin around the eyes was gently pulled back to make the eyeballs protrude slightly, and the eyeballs were grasped with forceps. Venous blood was collected into the corresponding EP tube. After standing for 30 minutes, the cells were centrifuged at 3000 rpm for 5 minutes. The supernatant was collected and transferred to a new EP tube and stored at -20 ℃.

[0144] After blood was collected from the orbital vein of the mice, they were immediately euthanized by dislocation. The skin and peritoneum were cut along the midline of the abdomen with scissors to expose the abdominal organs. The intestines were opened and the liver was removed to observe tumor formation, weigh, and photograph. The in situ tumor mass, liver, lungs, kidneys, etc., were harvested and observed with the naked eye. Fresh tissue samples were collected to extract RNA and proteins to detect the expression of relevant genes.

[0145] 1.4.4 Detection of Biochemical Indicators

[0146] The serum obtained by centrifugation was used to detect the following serum biochemical indicators in mice using a Beckman AU5800 biochemical analyzer: alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), and α-L-fucosidase (AFU).

[0147] 1.4.5 Statistical Methods

[0148] All experimental data are expressed as averages ( ). Mean ± standard deviation (SD) indicates that each experiment was independently repeated at least 3 times. Statistical comparisons between two groups were performed using t-tests, and comparisons among multiple groups were performed using one-way ANOVA. *P<0.05 and **P<0.01 indicate that the differences between groups are statistically significant.

[0149] Experiment 2

[0150] 2. Experimental Instruments and Materials

[0151] 2.1 Experimental Apparatus

[0152] Table 3 Experimental Instruments

[0153]

[0154] 2.2 Experimental Reagents

[0155] Table 4 Experimental Reagents

[0156]

[0157] Table 5 Antibody Information

[0158]

[0159] 2.3 Experimental Methods

[0160] 2.3.1 Cell Culture

[0161] Human HepG2 and Huh7 hepatocellular carcinoma cells were used. Human HepG2 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, while human Huh7 cells were cultured in DMEM medium containing the same antibiotics. Both cultures were incubated at 37°C in a 5% CO2 cell culture incubator. When the fusion rate of HepG2 and Huh7 cells reached over 80%, the cells were digested with 0.25% trypsin. The cells were then passaged at a 1:2 ratio for subsequent experiments.

[0162] 2.3.2 Extraction of total cellular protein

[0163] HepG2 and Huh7 liver cancer cells in logarithmic growth phase 1×10 6Cells were seeded in six-well plates, designated as a control group and an experimental group, and allowed to adhere. The control group received equal volumes of untreated MEM basal medium and DMEM medium, while the experimental groups received low (25 µmol / L), medium (50 µmol / L), and high (100 µmol / L) concentrations of TF-3 (…). Figure 2 The cells were cultured in the appropriate basal medium and incubated for 24 h. After 24 h, the cells were washed three times with PBS, the solution was discarded and aspirated. 100 µL of a 100:1:1 ratio of RIPA lysis buffer:PMSF:phosphatase inhibitor was added to each well, and the cells were incubated at 4 ℃ for 30 min. After cell lysis, HepG2 and Huh7 liver cancer cells treated with different concentrations of TF-3 for 24 h were collected in centrifuge tubes using a cell scraper. The tubes were subjected to three freeze-thaw cycles, sonicated for 10 s, centrifuged at 12000 rpm for 10 min at 4 ℃, and the supernatant was collected. A portion of the protein was analyzed using the BCA method to determine protein concentration. The corresponding volume of loading buffer was added, vortexed, and briefly centrifuged. The tubes were then boiled in a 95 ℃ metal bath for 10 min to denature the protein, aliquoted, and stored at -20 ℃.

[0164] 2.3.3 Western blot assay for proteins

[0165] (1) Prepare adhesive: Clean the adhesive glass plate (1.0 mm), dry the side of the glass plate that is in contact with the adhesive, assemble the adhesive preparation device, add the lower layer adhesive according to the ratio, and seal with anhydrous ethanol for 30 min. After the lower layer adhesive has completely solidified, remove the sealing liquid and wipe it clean with filter paper. Then add the upper layer adhesive, insert the comb and let it solidify for 30 min.

[0166] (2) Electrophoresis: After the upper layer solidifies, add electrophoresis solution and slowly pull out the comb. Add the sample to be tested and the marker to the lane one by one according to the normalized sample amount by vortex centrifugation. Run at 80 V for half an hour. After the marker separates, adjust the voltage to 150 V and stop the transfer when the voltage is 1 cm away from the bottom edge of the gel.

[0167] (3) Transfer: When electrophoresis is almost over, prepare the necessary transfer clamps, sponges, and filter paper in a special transfer tray, and pour in the pre-cooled transfer buffer. Activate the cut PVDF membrane by soaking it in methanol solution beforehand. After electrophoresis, remove the glass plate from the electrophoresis tank, pry it open, and cut the gel. Place each transfer sandwich in the following order: sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, ensuring air bubbles are removed and the sandwich is clamped tightly. Place it in the transfer tank, fill with transfer buffer, and perform the transfer at 300 A for 1 hour.

[0168] (4) Sealing: After the transfer is completed, take out the PVDF membrane, immerse it in the sealing solution (rapid sealing solution or BSA), and seal it at room temperature for 1 h on a horizontal shaker.

[0169] (5) Incubation with primary antibody: After the blocking is completed, the blocking solution is recovered, and the corresponding primary antibody diluted at a ratio of 1:2000 is added. The mixture is then incubated overnight on a horizontal shaker at 4 ℃.

[0170] (6) Incubation with secondary antibody: On the second day, the primary antibody was recovered, and the PVDF membrane was washed with TBST 3 times for 5 min. Then, the corresponding secondary antibody of the same species (1:4000) was added and incubated on a horizontal shaker at room temperature for 1 h.

[0171] (7) Development and data processing: The secondary antibody was recovered, and the membrane was washed three times with TBST for 5 min each time. Then, chemiluminescent reagents A and B were added in a 1:1 ratio for development and band images were collected. Image-J image analysis software was used to analyze the gray values ​​of the protein bands and to perform quantitative analysis of the target protein.

[0172] 2.3.4 Data Statistics and Analysis

[0173] All experimental data were analyzed using GraphPad Prism 10 software, and the results are expressed as averages. Mean ± standard deviation (SD) indicates that each experiment was independently repeated at least three times. t-tests were used for statistical comparisons between groups; one-way ANOVA was used for comparisons among multiple groups. *P<0.05 and **P<0.01 indicate statistically significant differences.

[0174] 2.4 Results

[0175] 2.4.1 TF-3 intervention inhibited PCID2 protein expression in HepG2 and Huh7 hepatocellular carcinoma cells.

[0176] To further explore the mechanism by which TF-3 intervention improves the malignant phenotype of hepatocellular carcinoma cells, this study used Western blotting to detect the changes in PCID2 protein expression levels in HepG2 and Huh7 hepatocellular carcinoma cell lines after TF-3 intervention.

[0177] After treating HepG2 hepatocellular carcinoma cells with TF-3 at concentrations of 25, 50, and 100 μmol / L for 24 h, the expression level of PCID2 protein in HepG2 hepatocellular carcinoma cells was found to be significantly decreased in a concentration-dependent manner compared with the control group, with increasing drug concentration (P<0.01). Figure 12 In the Huh7 cell line, compared with the control group, after 24 hours of TF-3 intervention, the expression level of PCID2 protein in Huh7 liver cancer cells was significantly downregulated with increasing intervention concentration (P<0.01). Figure 12The Western blot (WB) results showed that TF-3 intervention in HepG2 and Huh7 liver cancer cells yielded consistent results, downregulating the expression level of PCID2 protein in both HepG2 and Huh7 liver cancer cells.

[0178] 2.4.2 TF-3 intervention inhibits the expression of cell cycle-related proteins in HepG2 and Huh7 liver cancer cells.

[0179] After treating HepG2 hepatocellular carcinoma cells with TF-3 at concentrations of 25, 50, and 100 μmol / L for 24 h, the expression levels of Cyclin D1 and CDK6 proteins in HepG2 hepatocellular carcinoma cells were found to be significantly decreased in a concentration-dependent manner compared with the control group. Figure 13 In the Huh7 cell line, compared with the control group, after 24 hours of TF-3 intervention, the expression levels of cyclin D1 and CDK6 in Huh7 hepatocellular carcinoma cells were significantly downregulated with increasing intervention concentration (P<0.01). Figure 13 The Western blot results showed that TF-3 intervention in HepG2 and Huh7 liver cancer cells yielded consistent results, downregulating the expression levels of cyclin D1 and CDK6 in both HepG2 and Huh7 liver cancer cells.

[0180] 2.4.3 TF-3 intervention suppressed the protein expression levels of core factors in the PI3K / Akt / NF-κB signaling pathway in HepG2 and Huh7 hepatocellular carcinoma cells.

[0181] Compared with the control group, after treating HepG2 liver cancer cells with TF-3 at concentrations of 25, 50, and 100 μmol / L for 24 h, the expression levels of p-PI3K, p-AKT, and p-NF-κB proteins in HepG2 liver cancer cells were significantly reduced in a concentration-dependent manner with increasing intervention concentration (P<0.01). Figure 14 In the Huh7 cell line, compared with the control group, after 24 hours of TF-3 intervention, the expression of cell cycle proteins p-PI3K, p-AKT and p-NF-κB in Huh7 liver cancer cells was significantly reduced in a concentration-dependent manner with increasing intervention concentration (P<0.01). Figure 14 The Western blot (WB) results showed that TF-3 intervention in HepG2 and Huh7 liver cancer cells yielded consistent results, inhibiting the protein expression levels of core factors in the PI3K / AKT / NF-κB signaling pathway.

[0182] Example 1: Screening and Construction of PCID2 Inhibition Candidate Molecules Based on Existing Compound Libraries

[0183] In this embodiment, a preliminary screening was conducted from a small molecule compound library containing various aromatic compounds. The screening criteria were that the molecular structure simultaneously contained multiple hydroxyl substituents and at least one rigid cyclic skeleton. Through structural analysis, two representative candidate molecules were selected as research objects, whose molecular structures, in terms of spatial configuration and functional group distribution, possess the potential to bind to the PCID2 protein.

[0184] Further computer-aided analysis confirmed that the candidate molecule can spatially enter the functional binding region of PCID2, with hydroxyl groups forming stable hydrogen bonds with key protein residues, and the cyclic backbone matching the hydrophobic region of the protein. This synergistic structural feature provides a clear molecular basis for subsequent PCID2 functional inhibition.

[0185] Example 2: Verification of molecular docking and synergistic effect between candidate molecules and PCID2 protein

[0186] In this embodiment, molecular docking calculations are used to analyze the binding modes of the selected candidate molecules with the PCID2 protein. By constructing a three-dimensional structural model of the PCID2 protein, flexible docking calculations are performed on the candidate molecules to obtain their optimal binding conformation on the PCID2 surface.

[0187] Analysis revealed that the candidate molecule forms a multi-point synergistic network during binding, with multiple hydrogen bonds forming between the hydroxyl groups and key amino acid residues of PCID2. The ring structure is embedded in the protein's hydrophobic pocket region, significantly enhancing binding stability. This synergistic binding mode validates the candidate molecule's potential for inhibiting PCID2 activity at the molecular level.

[0188] Example 3: In vitro inhibitory effect of PCID2 inhibitors on the proliferation of hepatocellular carcinoma cells

[0189] In this embodiment, in vitro cultured cells derived from human hepatocellular carcinoma were used as the research model. Screened and validated candidate molecules were added to the cell culture system, and a control group was set up under the same culture conditions. The effects of the candidate molecules on cell proliferation behavior were systematically evaluated using cell viability and colony formation assays.

[0190] The experimental results showed that, compared with the control group, the proliferation rate of hepatocellular carcinoma cells in the treatment group was significantly reduced, and the number of colonies formed was significantly decreased. Simultaneously, a decrease in the expression level or functional activity of PCID2 protein was detected, indicating a direct correlation between the cell proliferation inhibition effect and the PCID2-suppressed state.

[0191] Example 4: Target Specificity Verification of PCID2 Inhibition

[0192] In this embodiment, to verify the target specificity of the inhibitory effect of the candidate molecules, PCID2-related signaling pathways were analyzed in treated hepatocellular carcinoma cells. The action pathways of the candidate molecules were evaluated by detecting changes in PCID2 protein levels and the status of related cell proliferation regulators.

[0193] The results showed that treatment with the candidate molecules significantly inhibited PCID2-related regulatory pathways, while cellular basal metabolic pathways not directly related to PCID2 remained largely unchanged. This indicates that the candidate molecules exert their anti-proliferative effect through a specific inhibitory mechanism targeting PCID2, rather than through non-specific cytotoxicity.

[0194] Example 5: Safety evaluation of PCID2 inhibitors at in vitro and in vivo levels

[0195] In this embodiment, the candidate molecule was applied to an established hepatocellular carcinoma animal model, and tumor growth changes were observed through continuous administration. The mice's condition was observed throughout the experimental period, and weight changes were recorded periodically, with comparisons made to a control group without medication. Results showed that no adverse effects such as lethargy or rough coat were observed in mice at different dosage intervention groups; TF-3 was safe for mice at the appropriate intervention dose and had little impact on weight. Macroscopic observation of the kidney and lung tissues of mice in each group revealed no significant abnormalities; HE staining of the liver and kidney tissues showed no significant difference compared to the control group. These results preliminarily indicate that TF-3 has no potential toxic effects on mouse tissues.

[0196] Serum biochemical parameters of mice in each group were measured. The results showed that, compared with the model group, only ALP decreased significantly after high-concentration intervention (P<0.05), while the changes in other parameters were not statistically significant (P>0.05). Compared with the control group, there were no significant differences in serum biochemical parameters of TF-3, suggesting that TF-3 has no significant toxic effects on the liver within the selected intervention concentration range.

[0197] In this embodiment, the candidate molecule was applied to normal human hepatocytes, and the activity of the normal human hepatocytes after intervention was detected. The results showed that the candidate molecule inhibited the activity of normal human hepatocytes in a dose-dependent manner; compared with the results of intervention on HepG2 liver cancer cells, the survival rate of normal hepatocytes was higher when the intervention dose of the candidate molecule was higher (100 μmol / L). Therefore, it can be considered that the candidate molecule has a certain degree of safety for normal hepatocytes.

[0198] The comprehensive evaluation results demonstrate that the candidate molecules possess both biological activity and a sound safety profile, providing support for future applications.

[0199] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A PCID2 inhibitor molecule, characterized in that, The PCID2 inhibitor molecule can specifically bind to the functional binding site of the PCID2 protein; The inhibitor molecule comprises multiple hydroxyl substituents and a rigid cyclic backbone structure, wherein: Polyhydroxy substituents are used to form a stable hydrogen bond network with key amino acid residues of PCID2; The rigid ring-shaped framework is used to form a spatially complementary hydrophobic interaction with the PCID2 hydrophobic pocket.

2. The PCID2 inhibitor molecule as described in claim 1, characterized in that, The inhibitor molecules can significantly reduce the functional activity or effective expression level of PCID2 protein in cells.

3. The PCID2 inhibitor molecule as described in claim 1, characterized in that, The inhibitor molecules inhibit abnormal cell proliferation by interfering with PCID2-mediated signaling pathways related to cell proliferation regulation.

4. The PCID2 inhibitor molecule as described in claim 1, characterized in that, The inhibitor molecules were obtained from screening existing compound libraries and possess both polyhydroxy substitution structures and rigid cyclic skeleton structures.

5. The PCID2 inhibitor molecule as described in claim 4, characterized in that, The inhibitor molecule was verified by molecular docking calculations to determine its conformational stability and key interaction sites with the PCID2 protein.

6. Use of the PCID2 inhibitor molecule according to any one of claims 1 to 5 in the preparation of a medicament or bioactive composition for regulating PCID2-related cell proliferation signaling pathways.

7. The use as described in claim 6, characterized in that, The drug or bioactive composition is used to inhibit abnormal cell proliferation associated with high PCID2 expression.

8. A bioactive composition, characterized in that, Includes the PCID2 inhibitor molecule as described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

9. The bioactive composition according to claim 8, characterized in that, The composition exhibited good biocompatibility and acceptable in vivo metabolic behavior in in vitro human normal liver cells and animal models.

10. The bioactive composition according to claim 8 or 9, characterized in that, The composition exhibited a biological effect of inhibiting abnormal cell proliferation in a PCID2-overexpression-associated hepatocellular carcinoma model.

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