Application of a small molecule compound targeting PGK1 K131 in the treatment of intrahepatic cholangiocarcinoma

Abstract

This invention provides the application of a small molecule compound targeting PGK1 K131 in the treatment of intrahepatic cholangiocarcinoma. It is the first time that the lactation modification site of PGK1 K131 has been identified as an effective new target for iCCA. The first highly active small molecule targeting this site, ZINC000150392143, has been discovered and verified. It can inhibit the proliferation, migration and invasion of iCCA cells in vitro and significantly inhibit the growth of in situ liver tumors in vivo. This provides a new broad-spectrum anti-tumor strategy to overcome the limitations of existing iCCA therapeutic targets.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically to the application of a small molecule compound targeting PGK1 K131 in the treatment of intrahepatic cholangiocarcinoma. Background Technology

[0002] Diagnostic and therapeutic technologies related to intrahepatic cholangiocarcinoma (iCCA) are primarily applied in the field of oncology within clinical medicine, specifically focusing on the diagnosis and treatment of malignant liver tumors. In medical practice, the liver, as a core metabolic organ, has always been a key focus and challenge in the clinical diagnosis and treatment of primary malignant tumors. iCCA, as one of the major types of primary malignant liver tumors, has become a significant threat to human health. From an application perspective, these technologies broadly cover multiple stages, including treatment planning after clinical diagnosis, palliative care for advanced-stage patients, postoperative adjuvant therapy, and clinical research on novel therapeutic drugs. iCCA originates from the epithelial cells of the secondary bile ducts and their branches within the liver and is a type of adenocarcinoma. Due to its insidious location and atypical early clinical symptoms, most patients are diagnosed at an advanced stage, significantly increasing the difficulty of clinical treatment. With the deepening application of molecular biology technology in the field of oncology, the understanding of the molecular pathology of iCCA is constantly improving. Targeted therapy and immunotherapy have gradually become important research directions in this field. Among them, small molecule compounds have become the core strategy for developing new iCCA therapeutic drugs due to their unique advantages such as high oral bioavailability, precise targeting of intracellular targets, and easy optimization of drug efficacy through chemical modification. They are widely used in drug development and clinical applications targeting specific molecular targets of iCCA.

[0003] In the clinical treatment of iCCA, several critical issues urgently need to be addressed, severely impacting patient outcomes and survival. First, the difficulty in early diagnosis leads to delayed treatment. Early iCCA often presents with no obvious specific symptoms, typically only mild abdominal discomfort and fatigue, which are easily overlooked. By the time most patients are diagnosed, the optimal time for surgical resection has passed, and surgery is currently the only potentially curative treatment. This situation directly results in a poor overall prognosis. Second, traditional treatments have limited efficacy. For inoperable advanced iCCA patients, commonly used radiotherapy and chemotherapy regimens are ineffective in suppressing tumor growth, failing to effectively control disease progression, significantly prolong survival, or improve quality of life. Furthermore, iCCA exhibits significant metabolic reprogramming characteristics, a crucial driver of tumor progression. Specifically, even under adequate oxygen conditions, tumor cells tend to employ aerobic glycolysis (the Warburg effect), converting large amounts of glucose into lactic acid. This abnormal metabolism not only provides ample substances and energy for the rapid proliferation of cancer cells, but also actively reshapes the tumor microenvironment, causing it to exhibit immunosuppression and pro-fibrotic states, further promoting tumor invasion and metastasis, while also increasing the difficulty of treatment. Finally, significant individual differences exist among patients, and the molecular subtypes of tumors are complex and diverse, making it difficult for a single treatment regimen to be applicable to all patients, and the implementation of precision medicine faces enormous challenges.

[0004] To address the aforementioned challenges in iCCA treatment, existing technologies have undertaken a series of targeted solutions, achieving breakthroughs, particularly in the field of precision medicine. With in-depth research into the molecular pathological mechanisms of iCCA, targeted therapy has become a crucial direction for overcoming the limitations of traditional radiotherapy and chemotherapy. Among these, small molecule targeted therapy based on specific molecular subtypes is currently a research and application hotspot. Specifically, existing small molecule targeted therapy technologies mainly revolve around two key targets: one is inhibitors targeting fibroblast growth factor receptor (FGFR) fusions / rearrangements. These drugs are specifically suitable for iCCA patients with specific gene alterations such as FGFR2 fusions, blocking tumor cell proliferation and survival by precisely inhibiting FGFR-related signaling pathways; the other is inhibitors targeting isocitrate dehydrogenase 1 (IDH1) mutations, primarily used to treat iCCA patients carrying IDH1 hotspot mutations, intervening in abnormal metabolic processes of tumor cells by inhibiting the activity of mutant IDH1. The successful development and clinical application of these targeted drugs mark the formal entry of iCCA treatment into the precision medicine stage. They have brought significant clinical benefits to certain subgroups of patients, effectively prolonging their survival and reducing the severe adverse reactions associated with traditional radiotherapy and chemotherapy, thus improving the patient experience. Furthermore, researchers have also explored the metabolic reprogramming characteristics of iCCA, using metabolic enzymes as potential therapeutic targets to inhibit tumor progression by intervening in abnormal metabolic pathways of tumor cells. The development and application of IDH1 inhibitors is a significant achievement in this direction.

[0005] While existing small-molecule targeted therapies have brought new hope to the treatment of iCCA, many key issues remain unresolved, failing to meet the clinical treatment needs of a large number of patients. First, the treatment coverage is extremely limited. Currently approved FGFR inhibitors and IDH1 inhibitors are only applicable to patients carrying specific gene mutations, but these patients represent a small proportion of the overall iCCA population. The vast majority of patients who do not carry these known druggable target mutations cannot benefit from existing targeted therapies and can only rely on traditional treatments with limited efficacy, leaving a significant unmet clinical need. Second, drug resistance is widespread and difficult to overcome. Acquired drug resistance is a common clinical challenge in iCCA targeted therapy, with complex and diverse mechanisms, including secondary mutations of the target itself (preventing drugs from effectively binding to the target) and activation of bypass signaling pathways (tumor cells bypassing the inhibited target and continuing to proliferate through other signaling pathways). Existing drugs cannot effectively address these resistance mechanisms, leading to easy tumor recurrence and progression in the later stages of treatment, severely affecting the durability of treatment effects. Finally, interventions targeting the core metabolic pathways of iCCA are significantly insufficient. Currently approved IDH1 inhibitors only target specific mutant IDH1 cells, failing to broadly and effectively intervene in the core glucose metabolism reprogramming pathways (such as aerobic glycolysis) prevalent in iCCA. This prevents the fundamental blocking of the core mechanism by which most iCCA cells proliferate through abnormal metabolism. In summary, current technologies still cannot overcome the core challenges of limited applicability, significant drug resistance, and incomplete metabolic intervention in iCCA treatment. There is an urgent need to develop novel therapeutic technologies and drugs that target the prevalent metabolic pathways of iCCA, cover a wider patient population, and effectively overcome drug resistance. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the application of a small molecule compound targeting PGK1 K131 in the treatment of intrahepatic cholangiocarcinoma. For the first time, it clearly identifies the lactation modification site of PGK1 K131 as an effective new target for iCCA. The invention also discovers and verifies the first highly active small molecule targeting this site, ZINC000150392143, which can inhibit the proliferation, migration, and invasion of iCCA cells in vitro and significantly inhibit the growth of in situ liver tumors in vivo. This provides a new broad-spectrum anti-tumor strategy to overcome the limitations of existing iCCA therapeutic targets.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the application of phosphoglycerate kinase 1 modified by lactation of lysine at position 131 as a target for screening drugs that inhibit intrahepatic cholangiocarcinoma.

[0008] The present invention also provides a target for phosphoglycerate kinase 1 modified by lactation of lysine at position 131, and its use in the preparation of pharmaceutical compositions for inhibiting intrahepatic cholangiocarcinoma.

[0009] Furthermore, the target includes a small molecule compound with ZINC number ZINC000150392143, with the following structural formula:

[0011] Furthermore, the target can significantly inhibit, in vitro, cell proliferation dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; cell migration dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; and cell invasion dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131.

[0012] Furthermore, the target significantly inhibits the growth of in situ liver tumors in vivo.

[0013] This invention also provides the use of a small molecule compound in the preparation of a drug for treating intrahepatic cholangiocarcinoma, wherein the small molecule compound has the ZINC number ZINC000150392143 and the structural formula is as follows:

[0015] Furthermore, the small molecule compound can specifically target phosphoglycerate kinase 1 in intrahepatic cholangiocarcinoma cells, and its targeting action is the lactation modification site of lysine at position 131 of PGK1.

[0016] Furthermore, the lactation modification of lysine at position 131 of PGK1 is achieved by adding a lactic acid group to the lysine side chain.

[0017] Furthermore, the small molecule compound can significantly inhibit, in vitro, cell proliferation dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; cell migration dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; and cell invasion dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131.

[0018] Furthermore, the small molecule compound significantly inhibits the growth of in situ liver tumors in vivo.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: Current iCCA treatment targets largely rely on gene mutations such as FGFR2 and IDH1, but the incidence of these mutations in iCCA patients is limited, resulting in most patients not receiving effective targeted therapy. The PGK1 K131R target established in this invention addresses this issue because PGK1 protein is widely highly expressed in iCCA cells, and its pro-cancer function depends on lactation modification at the K131 site. This target can cover the vast majority of iCCA patients, significantly expanding the applicability of iCCA treatment strategies and solving the core problem of insufficient patient coverage in existing technologies. Furthermore, this invention reveals a novel molecular mechanism by which PGK1 mediates the development and progression of iCCA, expanding the theoretical understanding of tumor metabolic targeted therapy. This invention, for the first time, demonstrates through a PGK1 knockdown and K131 site complementation cell model that the oncogenic function of PGK1 does not depend on its classic glycolytic activity, but rather on the lactation modification of its lysine residue at position 131. This discovery overturns the traditional understanding of the PGK1 oncogenic mechanism, providing new theoretical support for the metabolic-driven pathogenesis of iCCA and opening up new directions for targeted therapy research in the field of tumor metabolism. This invention uses PGK1 K131R as a drug screening target. Through mature technologies such as molecular docking and virtual screening, it can precisely locate potential inhibitors that specifically bind to this target, effectively reducing the blind spots in drug development, lowering development costs, and shortening the development cycle. It provides a core tool and clear direction for the efficient development of iCCA targeted drugs.

[0020] The target compound in this invention specifically recognizes and binds to the PGK1 K131R site, precisely intervening in the pro-cancer function mediated by lactation modification at this site without affecting the normal glycolytic activity of the PGK1 protein. This avoids interference with normal cellular metabolic processes, significantly reduces off-target effects during targeted therapy, minimizes drug damage to normal tissues, and improves the therapeutic safety and tolerability of the drug composition. By specifically binding to PGK1 K131R, this target compound effectively inhibits the proliferation, migration, and invasion of iCCA cells, and significantly inhibits the growth of iCCA in situ tumors in vivo, exhibiting a clear and stable anti-tumor effect. This provides a novel drug development direction and potential therapeutic tool for the clinical treatment of iCCA, filling a gap in existing targeted therapies. Since the PGK1 K131R target of this drug is prevalent in iCCA, its applicable population is not limited by gene mutation status such as FGFR2 and IDH1. It can cover the vast majority of iCCA patients who cannot benefit from existing targeted therapies, effectively solving the clinical pain point of the narrow applicable population of existing treatment options and providing a feasible technical path for broad-spectrum targeted therapy of iCCA.

[0021] This invention, through precise molecular docking, virtual screening, and in vitro and in vivo experimental verification, has for the first time discovered and confirmed that ZINC000150392143 is a highly active small molecule compound that specifically targets PGK1 K131R. Its successful discovery fills the drug gap in the iCCA field targeting PGK1 K131R, providing a core lead compound for the development of subsequent novel targeted drugs. This small molecule compound, ZINC000150392143, binds with high affinity to the K131 site of the PGK1 protein and the surrounding lactylation modification pocket. This pocket is a key region for lactate group binding and function. This highly efficient and specific binding mode is the core reason why it can precisely inhibit lactation modification at the K131 site and exert significant anti-tumor activity, further ensuring its high activity and specificity advantages as a candidate drug. This small molecule compound, validated by in vitro CCK-8 assays, plate colony formation assays, cell scratch assays, and Transwell assays, specifically inhibits the proliferation, migration, and invasion of PGK1 K131R-dependent iCCA cells. Furthermore, nude mouse iCCA orthotopic xenograft experiments further confirmed that the compound significantly inhibits the volume and weight increase of orthotopic tumors in vivo. These in vitro and in vivo experiments form a complete chain of evidence for efficacy, fully demonstrating the certainty and stability of its anti-tumor effect, providing solid experimental support for subsequent clinical translational applications. As a candidate drug for iCCA targeted therapy, this small molecule compound possesses good drug-like characteristics and drug development potential, and is expected to be further optimized in subsequent drug development to become a novel targeted drug for clinical use. Its application can effectively address the limitations of existing iCCA treatment options and the narrow applicable population, especially providing a new treatment option for iCCA patients without FGFR2 or IDH1 mutations. It can significantly improve patient prognosis, reduce patient suffering and medical burden, and has significant clinical application value and social significance.

[0022] Overall, this invention not only deepens the understanding of the pathogenesis of iCCA in theory and breaks through the cognitive limitations of traditional tumor metabolic targeted therapy, but also solves the key technical defects in the existing iCCA treatment field in practice. It provides a brand-new strategy and tool for the targeted therapy of iCCA, accelerates the research and development process of new targeted drugs for iCCA, and has important scientific value, technological innovation and clinical application prospects. It is expected to promote technological innovation in the field of iCCA treatment. Attached Figure Description

[0023] Figure 1 The molecular structure diagram of the small molecule ZINC000150392143 is shown.

[0024] Figure 2Figures show the results of the CCK-8 assay (A) and the plate colony formation assay (B), where: CON, control group; DMSO, solvent control; ZINC000150392143, target small molecule compound; ShPGK1, cell line with stable PGK1 knockdown; WT, wild type; K131R, PGK1 mutant with lysine mutated to arginine at position 131. OD value (450nm) represents cell proliferation; Colony Numbers represent colony formation ability; ns indicates no statistical significance.

[0025] Figure 3 The figures show the results of the scratch test (A) and the Transwell test (B). Invasion / Migration refer to the Transwell invasion and migration tests, respectively. OH / 24H represent the treatment start (0 hours) and 24 hours after treatment, respectively. ns indicates that the difference between groups was not statistically significant.

[0026] Figure 4 This is a graph showing the results of an orthotopic tumor transplantation experiment in mice. Where: Tumor Volume: Tumor volume (mm) 3 Tumor Weight: The actual weight (g) of the tumor removed at the end of the experiment. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] This invention addresses the key technical deficiency in the field of iCCA, namely the limitation of therapeutic targets (such as the limited proportion of patients with FGFR2 / IDH1 mutations), and proposes a new solution: to develop a broad-spectrum anti-tumor strategy by targeting the core metabolic protein of iCCA, which is widely highly expressed in cells—intracellular phosphoglycerate kinase 1 (PGK1). To achieve this goal, this invention conducted a series of related experiments, the experimental procedure of which is as follows: First, protein structure preparation was performed. The PGK1 protein structure was obtained from the Uniprot database, and the K131 (Lys131) site was modified by in vitro lactation using UCSF Chimera to simulate the post-translational modification state, resulting in phosphoglycerate kinase 1 (PGK1 K131) modified by lactation of lysine at position 131. Subsequently, binding site analysis was performed. The modified protein was analyzed using SiteMap to identify potential drug binding pockets. Next, molecular docking and screening were carried out. Based on the above binding pockets, the ZINC20 drug molecule library (containing 2.39 million small molecules) was batch-virtually screened using the Autodock 1.2.0 tool. In the docking settings, the maximum number of search conformations was set to 10,000, and a genetic algorithm was used for conformation sampling and scoring. Finally, the results were output and verified. The molecular conformations were sorted according to the docking scores, and the candidate molecules corresponding to the optimal conformations were selected. These calculated and screened specific candidates will be submitted to subsequent experiments for biological verification.

[0029] Meanwhile, this invention is the first to demonstrate in iCCA that the oncogenic function of PGK1 depends on the lactation modification of its 131st lysine residue (K131), rather than being mediated solely by its classical glycolytic activity. This conclusion was verified by constructing a PGK1 knockdown and K131 site complementation cell model, and by using in vitro analyses such as CCK-8 assays, monoclonal antibody assays, cell scratch assays, and Transwell assays, combined with an in vivo experiment using a nude mouse intrahepatic cholangiocarcinoma orthotopic xenograft model. This provides a direct and innovative theoretical basis for developing novel inhibitors targeting this site.

[0030] Furthermore, this invention discovered and validated the first highly active small molecule compound targeting PGK1 K131, ZINC000150392143. This compound specifically inhibits PGK1 K131-dependent cell proliferation, migration, and invasion in vitro, and significantly inhibits the growth of in situ liver tumors in vivo, fully demonstrating its potential as a candidate drug. Specific experiments are as follows: 1. Experimental equipment and reagents 1.1 Cell lines, experimental animals, and small molecule compounds used This invention uses the intrahepatic bile duct cell line RBE (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and NOD.Cg-Prkdc 0cid Il2rg tm ¹ sug / JicCrl immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used to construct an orthotopic xenograft model of intrahepatic cholangiocarcinoma. The PGK1 knockdown vector (shRNA sequence 5'-CCGGCTGACAAGTTTGATGAGAATGCTCGAGCATTCTCATCAAACTTGTCAGTTTTTG-3'), the PGK1 overexpression vector (pcDNA3.1-PGK1-3×FLAG-Neo), and its 131st site mutant plasmid (pcDNA3.1-PGK1-3×FLAG-Neo-K131R) used in the experiment were all designed and constructed by Wuhan Paiwei Biotechnology Co., Ltd. Specifically, the 131st site mutant plasmid involves lactation modification of the 131st lysine residue of the PGK1 protein, achieved by adding a lactic acid group (-CO-CH(OH)-CH3) to the lysine side chain.

[0031] The small molecule compound ZINC000150392143 was provided by Vitas-M Laboratory, and its structure is as follows: Figure 1 As shown.

[0032] 1.2 Main Experimental Instruments and Equipment

[0033] 1.3 Main Experimental Reagents

[0034] 2. In vivo functional experiments 2.1 CCK-8 assay for cell proliferation 1) Cells in the logarithmic growth phase were grouped and treated as follows: ShCON group (blank control group): transfected with a non-targeted control sequence shRNA. ShPGK1-CON group (knockdown control group): first transfected with shRNA targeting PGK1 to construct stable knockdown cells, then transfected with an empty vector plasmid; ShPGK1-PGK1 WT group (wild-type complementation group): in the above PGK1 knockdown cell lines, a plasmid expressing wild-type human PGK1 protein was transfected; ShPGK1-PGK1 K131R group (mutant complementation group): in the same PGK1 knockdown cell lines, a PGK1 mutant plasmid expressing lysine at position 131 (K131R) was transfected. After transfection and culture for the specified time, cells in all groups were trypsinized, resuspended, counted, and the cell concentration was uniformly adjusted to 1×10⁻⁶ cells using complete culture medium. 4 cells / mL.

[0035] 2) Seed the cell suspension of each group at 200 µL / well in 96-well plates, with 5 replicates per group. After seeding, add compound ZINC000150392143 to each well to a final concentration of 100 mM. Plate five identical 96-well plates in parallel, corresponding to the detection time points after 6, 24, 48, 72, and 96 hours of culture. Incubate all plates at 37°C in a 5% CO2 incubator. Observe the cell status daily and change the culture medium as needed.

[0036] 3) Before each test, replace 100µL of fresh culture medium in each well and add 10mL of CCK-8 solution. After incubation for 2 hours, measure the absorbance (OD value) of each well at 490nm using an Epoch microplate reader.

[0037] 2.2 Plate colony formation experiment 1) Cell Seeding and Treatment: Take the single-cell suspensions from each group and seed them into 6-well plates at a density of 300-400 cells per well. Add 2 mL of 1640 medium to each well. Gently shake to disperse the cells evenly, then add compound ZINC000150392143 to each well to a final concentration of 100 mM. The culture plates are then placed in a 37°C, 5% CO2 incubator and cultured for 2-3 weeks, periodically replacing the medium with fresh medium containing the same concentration of the drug.

[0038] 2) Clonal fixation and staining: When clonal formation is visible to the naked eye, terminate the culture. Discard the culture medium and wash the cells three times with PBS. Add 4% paraformaldehyde solution to each well and fix for 15 minutes at room temperature. After discarding the fixative, add an appropriate amount of crystal violet staining solution and stain for 10-30 minutes.

[0039] 3) Clone counting: After staining, rinse off excess stain with running water and air dry at room temperature. Invert the dried culture plate and take an image of the entire culture dish. Use ImageJ software to analyze the clone count.

[0040] 2.3 Scratch Healing Experiment 1) Cell Seeding and Treatment: Take the single-cell suspensions from each group and seed them into 6-well plates at a density of 300-400 cells per well. Add 2 mL of 1640 medium to each well. After the cells have grown to 90%-100% confluent monolayers, use a 10 mL sterile pipette tip to make a straight line in the center of each well, perpendicular to the bottom of the plate. Gently wash three times with PBS to remove detached cells, and then add 2 mL of medium to each well.

[0041] 2) The treatment group was treated with compound ZINC000150392143 to a final concentration of 100 mM, while the control group received an equal volume of solvent. The culture plates were incubated at 37°C in a 5% CO2 incubator. Every 6 hours after the scratching, the scratch width was recorded using a phase-contrast microscope at the same location. The scratch area was measured using ImageJ software, and the cell migration rate or healing percentage for each group was calculated.

[0042] 2.4 Transwell Experiment 1.1 Cell invasion assay The test was performed using a Transwell chamber (8.0 mm aperture).

[0043] 1) Matrigel plating: Melt Matrigel at 4°C before the experiment. Before use, dilute with pre-cooled serum-free 1640 medium at a volume ratio of 1:8 (final concentration 1 mg / mL). Take 100 mL of the diluted solution and add it vertically to the center of the bottom of the upper chamber of the Transwell chamber. Let it stand at room temperature to allow it to polymerize and form a gel.

[0044] 2) Basement membrane hydration: Discard excess liquid, add 50 mL of serum-free 1640 medium containing 10 g / L BSA to each chamber, and incubate at 37°C for 5 minutes to hydrate the basement membrane.

[0045] 3) Cell preparation and seeding: Collect cells from each group, resuspend them in serum-free 1640 medium, and starve them at 37°C for 12 hours. Adjust the cell density to 5 × 10⁻⁶ cells / year. 5 cells / mL. 200 mL of cell suspension was seeded into the hydrated upper chamber of a Transwell. The treatment group received compound ZINC000150392143 to a final concentration of 100 mM in the upper chamber culture medium, while the control group received an equal volume of solvent.

[0046] 4) Culture and induction: Add 600 mL of 1640 complete medium containing 10% FBS as a chemokine to the lower chamber of a 24-well plate. Carefully place the Transwell chamber into the well plate and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0047] 5) Staining and Counting: After culture, remove the chamber and gently rinse with PBS. Carefully wipe away any unmigrated cells from the inner layer of the upper chamber membrane with a moistened cotton swab. Fix the chamber in 95% ethanol for 5 minutes, air dry, and then stain with crystal violet solution for 10-30 minutes. Wash away excess stain with PBS and air dry. Under a 200x inverted microscope, randomly select 8 fields of view for photography and count the number of cells that have passed through the membrane. Set up 3 replicates for each group, and repeat the experiment independently 3 times. Take the average value of the results.

[0048] 1.2 Cell migration experiment The migration assay is basically the same as the invasion assay, except that Matrigel is not used in the upper chamber of the Transwell assay. All other cell treatment, seeding, culture, staining and counting methods are the same.

[0049] 3. In vivo antitumor drug efficacy experiment This invention uses a mouse liver orthotopic tumor model to evaluate the in vivo antitumor activity of ZINC000150392143. NOD.Cg-Prkdcscid Il2rgtm1Sug / JicCrl (NOG) mice were selected, and the tumor was treated by intrahepatic orthotopic injection of 5 × 10⁻⁶ ZINC000150392143. 6 A tumor model was established using RBE cells. Approximately 14 days after inoculation (after tumor formation), mice were randomly divided into a model control group (DMSO solvent, 5 mg / kg) and a drug treatment group (ZINC000150392143, 5 mg / kg), with 6-8 mice in each group. Both groups received daily intraperitoneal injections for 3 weeks. After drug administration, mice were euthanized by carbon dioxide inhalation, and the livers were completely dissected and weighed. Tumor tissue was isolated, and its long axis (L) and short axis (W) were measured using the formula V = (L × W). 2 The tumor volume was calculated as () / 2. The tumor volume and weight of mice in the model control group and the drug treatment group were compared as core evaluation indicators.

[0050] Figure 2The results of the CCK-8 proliferation assay (A) and plate colony formation assay (B) clearly demonstrate the significant inhibitory effect of the small molecule compound ZINC000150392143 on the proliferation of intrahepatic cholangiocarcinoma cells. Specific results are analyzed below: In the CCK-8 assay (A), the control group cells maintained high proliferative activity at different culture time points (e.g., 24h, 48h, 72h), with corresponding absorbance (OD values) continuously increasing, indicating good cell proliferation. However, the experimental group treated with the small molecule ZINC000150392143 showed significantly lower OD values ​​at each time point than the control group, and the difference in proliferation between the two groups became increasingly pronounced with prolonged culture time. Statistical analysis showed a statistically significant difference (P<0.05), suggesting that this small molecule can inhibit the proliferative activity of iCCA cells in a time-dependent manner. In the plate colony formation assay (B), the control group cells formed numerous, large, and morphologically complete clonal colonies, exhibiting high colony formation efficiency. In the experimental group, after treatment with the small molecule, colony formation ability significantly decreased; microscopic examination revealed a marked reduction in the number and size of clones, with some clones exhibiting incomplete morphology. The colony formation efficiency was only about 50% of the control group, a statistically significant difference. This indicates that the small molecule effectively weakens the long-term proliferative potential and colony formation ability of iCCA cells. These in vitro proliferation assay results echo the core mechanism of small molecule-targeted inhibition of PGK1-K131R described earlier, and together with the subsequent migration and invasion assay results, corroborate that the small molecule ZINC000150392143 can significantly inhibit the malignant biological behavior of iCCA cells by targeting PGK1-K131R, providing solid in vitro experimental support for its potential as a targeted anti-iCCA drug candidate.

[0051] Figure 3The results of the cell scratch assay (A) and Transwell invasion assay (B) further confirmed the anti-intrahepatic cholangiocarcinoma activity of the small molecule compound ZINC000150392143. Specific results are as follows: In the scratch assay (A), the control group cells healed rapidly after a certain culture time, exhibiting strong cell migration ability. However, the experimental group treated with ZINC000150392143 showed a significantly slower scratch healing rate, with the healing rate only about half that of the control group (40% vs 83%). The remaining scratch width was significantly greater than that of the control group. Statistical analysis showed a statistically significant difference between the two groups (P<0.05), indicating that this small molecule can effectively inhibit the migration ability of iCCA cells. In the Transwell assay, we evaluated the motility of iCCA cells through invasion (upper chamber lined with matrix gel) and migration (without matrix gel) experiments. As shown in result (B), in the control group, a large number of cells could penetrate the matrix gel (invasion) or microporous membrane (migration) and adhere to the lower chamber; however, after treatment with the small molecule ZINC000150392143, the number of cells adhering to the lower chamber was significantly reduced in both invasion and migration experiments. Quantitative analysis showed that the invasion and migration rate of the experimental group decreased by approximately 50% compared to the control group, and the difference was statistically significant. This indicates that the small molecule can not only effectively inhibit the invasive ability of iCCA cells but also significantly weaken their migration ability. The above in vitro experimental results echo the core mechanism of the small molecule targeting and inhibiting PGK1-K131R and exerting its anti-iCCA effect described above, further verifying the inhibitory effect of the small molecule ZINC000150392143 on the malignant biological behavior (migration and invasion) of iCCA cells, and providing in vitro experimental evidence for its potential as a candidate drug targeting PGK1-K131R.

[0052] Figure 4 The results of the nude mouse orthotopic intrahepatic cholangiocarcinoma xenograft experiment further validated the antitumor activity of the small molecule compound ZINC000150392143 at the in vivo level. The experiment included a DMSO solvent control group and a ZINC000150392143 treatment group. After in vivo administration, the control group showed large and numerous in situ tumor nodules in the liver, with well-developed morphology and obvious invasive growth characteristics. In contrast, the ZINC000150392143 treatment group showed significantly reduced tumor nodule volume and number, and tumor proliferation was significantly inhibited. Quantitative analysis of tumor volume and weight showed that the tumor volume in the ZINC000150392143 treatment group was only about 1 / 3 to 1 / 2 of that in the control group, and the tumor weight was also significantly reduced. The difference between the groups was statistically significant (P<0.05), clearly demonstrating that this small molecule can effectively inhibit the growth of iCCA orthotopic tumors in vivo.

[0053] In summary, the series of experiments conducted in vitro and in vivo in this invention constitute a complete chain of evidence, fully verifying that the small molecule compound ZINC000150392143 exerts its anti-intrahepatic cholangiocarcinoma effect by targeting PGK1-K131R. Figure 2 The results showed that this small molecule could significantly inhibit the short-term proliferation activity and long-term colony formation ability of iCCA cells, with both the proliferation rate and colony formation efficiency being significantly lower than those of the control group. Figure 3 Further evidence confirms that this small molecule can effectively weaken the migration and invasion capabilities of iCCA cells, slow down the scratch healing rate, and significantly reduce the number of invading cells; Figure 4 In vivo experiments using nude mice with orthotopic tumors validated the aforementioned efficacy. The volume and weight of the orthotopic tumors in the treated group were significantly lower than those in the control group, and tumor proliferation was significantly inhibited. This series of experiments, from cell proliferation and malignant phenotype to in vivo tumorigenesis, forms a complete chain of evidence from in vitro functional verification to in vivo efficacy confirmation, providing solid experimental support for this small molecule as a candidate anti-iCCA drug targeting PGK1-K131R.

Claims

1. The application of phosphoglycerate kinase 1 modified by lactation of lysine at position 131 as a target for screening drugs that inhibit intrahepatic cholangiocarcinoma.

2. The use of a target of phosphoglycerate kinase 1 modified by lactation of lysine at position 131 in the preparation of a pharmaceutical composition for inhibiting intrahepatic cholangiocarcinoma.

3. The application according to claim 2, characterized in that, The target includes a small molecule compound with ZINC number ZINC000150392143, with the following structural formula: 。 4. The application according to claim 2, characterized in that, The target significantly inhibits, in vitro, cell proliferation dependent on phosphoglycerate kinase 1 modified by lactation at lysine position 131; cell migration dependent on phosphoglycerate kinase 1 modified by lactation at lysine position 131; and cell invasion dependent on phosphoglycerate kinase 1 modified by lactation at lysine position 131.

5. The application according to claim 2, characterized in that, The target significantly inhibits the growth of in situ liver tumors in vivo.

6. The application of small molecule compounds in the preparation of drugs for treating intrahepatic cholangiocarcinoma, characterized in that, The small molecule compound has the ZINC number ZINC000150392143 and its structural formula is as follows: 。 7. The application according to claim 6, characterized in that, The small molecule compound can specifically target phosphoglycerate kinase 1 in intrahepatic cholangiocarcinoma cells, and its targeting effect is the lactation modification site of lysine at position 131 of PGK1.

8. The application according to claim 7, characterized in that, The lactation modification of lysine at position 131 of PGK1 is achieved by adding a lactic acid group to the lysine side chain.

9. The application according to claim 6, characterized in that, The small molecule compound significantly inhibits, in vitro, cell proliferation dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; cell migration dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131; and cell invasion dependent on phosphoglycerate kinase 1 modified by lactation of lysine at position 131.

10. The application according to claim 6, characterized in that, The small molecule compound significantly inhibited the growth of in situ liver tumors in vivo.

Images