Application of lasalocid as transketolase inhibitor
Through molecular binding of Rasaloxi to TKT and in vitro experimental verification, the lack of drugs targeting TKT was solved, and effective treatment of cancers such as HCC was achieved, especially the inhibition and survival of HCC.
Patent Information
- Application Number
- CN202510690573.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-12
AI Technical Summary
Currently, there is a lack of drugs targeting transketolase (TKT) to treat highly aggressive hepatocellular carcinoma (HCC). TKT is highly expressed in cancer cells, promoting its metabolic and non-metabolic processes. Blocking TKT can improve sensitivity to sorafenib and prevent DNA damage. The existing drugs are not enough to effectively inhibit the development of HCC.
Lasaloxi was used as a transketolase inhibitor, and its binding to TKT was determined through molecular docking and molecular dynamics simulation, and a TKT knockdown HCC cell model was established. In vitro and in vitro and intravenous experiments were conducted to verify the anti-tumor effect of Lasaloxi. The dose was 15-60 mg/kg, which was used to prepare drugs to prevent or treat diseases related to high expression of TKT.
Lasaloxime significantly inhibits TKT function, reduces tumor volume, and improves mouse survival, showing therapeutic potential for tumors with high expression of TKT such as HCC, and has low cytotoxicity to normal.
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Figure CN120459082A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of lasaloxibal as a transketolase inhibitor. Background Art
[0002] Liver cancer is the second leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC), representing 80% of primary liver cancers, is a highly aggressive tumor originating from liver cells, commonly known as the "king of cancers." Its five-year survival rate is less than 10%. Effective treatments are urgently needed.
[0003] Increased glucose oxidation is a key characteristic of cancer cells, which favor glycolysis over mitochondrial oxidative phosphorylation to produce adenosine triphosphate (ATP). These heterogeneous metabolic processes are regulated by various enzymes. Transketolase (TKT) is a key, rate-limiting enzyme in the nonoxidative branch of the pentose phosphate pathway (PPP), producing over 85% of ribose-5-phosphate (R5P), an essential precursor for DNA and RNA biosynthesis and crucial for cancer cell proliferation. TKT mediates two reversible reactions in the PPP, dictating the direction of glucose metabolic flux in cancer cells, making it a promising target for tumor therapy. However, no drugs targeting TKT are currently available.
[0004] Studies have shown that TKT promotes HCC development through both metabolic and non-metabolic pathways, that TKT blockade sensitizes cells to sorafenib, and that TKT deficiency protects against DNA damage in the liver. Therefore, pharmacological inhibition of TKT could hinder HCC development and enhance therapeutic efficacy.
[0005] In this study, we discovered that TKT is associated with the metastatic potential of HCC. TKT is highly expressed in liver cancer cells. We validated that a small molecule with TKT inhibitory activity inhibited HCC cell proliferation and migration in vitro and slowed HCC cell growth in vivo. Our findings will facilitate the development of small molecule TKT inhibitors and novel HCC therapies. Summary of the Invention
[0006] The present invention provides a use of lasalocid as a transketolase inhibitor and a use of lasalocid in the preparation of a medicament for preventing or treating TKT-related cancers and other diseases, thereby improving the therapeutic effect of cancer.
[0007] To achieve the above objectives, the present invention provides the following technical solutions: lasaloxib is used as a transketolase inhibitor. The lasaloxib is stably bound to transketolase via molecular docking, and lasaloxib acts on and regulates transketolase. Lasaloxib is also used to modulate TKT function and thereby inhibit tumor progression; the application environment is in cells and mice. Computer-aided drug design and virtual screening revealed that lasaloxib specifically binds to TKT. Binding of lasaloxib to TKT was confirmed using molecular docking, molecular dynamics, and cellular thermal shift assays.
[0008] Furthermore, TKT knockdown HCC cells were established to evaluate the effect of lasaloxib in specifically regulating TKT to inhibit tumor cells.
[0009] Furthermore, the in vivo evaluation of the anti-tumor effect of lasaloxib was conducted in cell transplanted mice.
[0010] Furthermore, the administration method of lasaloxib is intraperitoneal injection, but is not limited thereto.
[0011] Furthermore, the dosage of lasaloxib is 15 to 60 mg / kg. In addition, the present invention also provides the use of lasaloxib in the preparation of a drug for preventing and treating diseases related to high TKT expression.
[0012] Furthermore, they found that lasaloxib can bind to the TKT protein and inhibit its function, thereby having a good preventive and therapeutic effect on tumor models caused by its overexpression. This suggests that lasaloxib may be a drug for preventing or treating liver cancer and other tumor diseases with overexpression of TKT.
[0013] Furthermore, tumors with high TKT expression include, but are not limited to, hepatocellular carcinoma (HCC), breast cancer, ovarian cancer, esophageal cancer, lung cancer, and head and neck cancer, indicating a poor prognosis. Therefore, TKT is a potential therapeutic target for cancer treatment.
[0014] Furthermore, to verify that lasaloxib can significantly improve tumors through TKT, specifically by significantly reducing tumor size in transplanted mice, molecular biology studies have shown that lasaloxib can reverse glucose phosphate pentose metabolism and could serve as a prodrug for the development of anti-tumor drugs targeting the non-oxidative pentose phosphate pathway.
[0015] Furthermore, the dosage of lasaloxib is 15 to 60 mg / kg.
[0016] Furthermore, the drug includes lasaloximate and a pharmaceutically acceptable excipient or carrier.
[0017] The beneficial effects of the present invention are as follows: experiments have shown that lasaloxib can be used as a transketolase inhibitor, lasaloxib can bind to the TKT protein and inhibit its function, thereby inhibiting cancer cell cloning and migration. Mouse tumor-bearing experiments have found that lasaloxib can significantly reduce tumor volume and increase mouse survival; inhibiting TKT activity provides a new target for tumor treatment, and lasaloxib can be used as a potential drug basis for treating TKT-highly expressed tumors such as liver cancer and other cancers. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Molecular docking and dynamic simulation diagram of lasaloximate and TKT. Figure 1 A is a three-dimensional simulation diagram of the molecular docking of lasalocib and TKT (PDB: 6HAD), with lasalocib represented by a green line; Figure 1 B is the 2D diagram of the interaction between lasalocid and the active pocket of TKT; Figure 1 C is the RMSF value within the 20 ns molecular dynamics simulation time; Figure 1 E is the RMSD plot as a function of 20 ns molecular dynamics simulation time, where the x-axis shows the time in ns and the y-axis shows the RMSD; Figure 1 D is the non-bonded interaction monitoring of lasaloxibet-TKT during molecular dynamics simulation. The y-axis represents 1000 conformations, the x-axis represents the amino acid residues interacting with lasaloxibet, and red represents ligand interaction.
[0019] Figure 2 The graph shows the results of lasaloxib inhibiting the proliferation of three types of liver cancer cells at different concentration gradients, and sorafenib is the positive control. Figure 2 A is the HepG2 cell line, Figure 2 B is MHCC97H cell line, Figure 2 C is the HCCLM3 cell line.
[0020] Figure 3 The graph shows the results of different Lasalox inhibition of liver cancer cell proliferation (clone formation experiment), and Sorafenib is the positive control. Figure 3 A is the total result diagram, Figure 3 B is the result of 5uM concentration of lasalocid inhibiting the colony formation of HepG2 cell line. Figure 3 C is the result of 25uM concentration of lasalocid inhibiting the clone formation of MHCC97H cell line. Figure 3 D shows the results of 5uM concentration of lasalocid inhibiting the clone formation of HCCLM3 cell line.
[0021] Figure 4 Lasalox inhibits the migration of liver cancer cells (cell scratch test). Figure 4A is the cell scratch test to determine whether HepG2 cells were treated with 10 μM lasalocib and 20 μM sorafenib, MHCC97H cells were treated with 20 μM lasalocib and 40 μM sorafenib, and HCCLM3 cells were treated with 10 μM lasalocib and 20 μM sorafenib; Figure 4 B is a schematic diagram of cell proliferation over 24 hours. It can be seen from the figure that lasaloxi has an inhibitory effect on liver cancer cell migration that is similar to or even better than sorafenib.
[0022] Figure 5 Schematic diagram of CCK8 assay of cell survival rate of normal hepatocytes LO2 treated with different concentrations of lasaloxib and sorafenib at different times. As can be seen from the figure, the inhibitory effect of lasaloxib on hepatocyte proliferation is similar to that of sorafenib, and both are time-concentration dependent. The IC50 values of sorafenib at 24, 48, and 72 hours are 29.53, 10.20, and 12.23 μM, respectively, and the IC50 values of lasaloxib at 24, 48, and 72 hours are 24.74, 10.54, and 11.03 μM, respectively.
[0023] Figure 6 The cell heat shift method was used to verify whether lasaloxib binds to the TKT protein. As shown in the figure, compared with the control, lasaloxib destroyed the stability of the TKT protein as the temperature increased.
[0024] Figure 7 control group, sorafenib 30 mg·kg -1 and lasalocid 15, 30, and 60 mg kg -1 Schematic diagram of in vivo tumor growth photos.
[0025] Figure 8 This is a schematic diagram of the effect of lasaloxib on the growth of MHCC97H nude mouse xenograft tumors in different groups and time. As can be seen from the figure, the 8-day treatment cycle of 15 mg kg -1 There was no significant effect on the body weight of tumor-bearing mice.
[0026] Figure 9 The effect of lasaloxib on the growth of MHCC97H nude mouse xenograft tumor is shown in the histogram of tumor weight in different groups of mice. As can be seen from the figure, the 8-day treatment cycle of 15 mg kg -1 Inhibits tumor growth in tumor-bearing mice.
[0027] Figure 10 The figure shows the effect of lasaloxib on the growth of MHCC97H nude mouse xenograft tumors, and the relationship between the tumor volume and time in different groups of mice. As can be seen from the figure, the 8-day treatment cycle of 15 mg kg -1 Inhibit tumor volume in tumor-bearing mice with 30mg·kg -1 Same as Sorafenib. DETAILED DESCRIPTION
[0028] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.
[0029] Example 1: Prediction of affinity of lasalocid to TKT transketolase
[0030] 1. Molecular docking of lasaloxib and TKT transketolase
[0031] The molecular docking study between TKT transketolase protein crystal structure (PDB: 6HAD) and lasaloxi was performed using the CDOCKER module of Discovery Studio 2021 (DS, Accelrys Inc., San Diego, CA, USA). The TKT protein was optimized using the "Macromolecules" module. Lasaloxi was optimized using the "Prepare Ligands" and "Minimize Ligands" modules. During the docking process, the active site was defined with the ligand in the complex protein crystal structure as the center, and the radius of the active sphere was The CDOCKER calculation was performed without changing the default settings. The results showed that in the binding of the original ligand thiamine pyrophosphate to TKT, ASN185, LYS244, GLY156, and GLU157 all formed H-bonds with the phosphate group of the ligand. The RMSD between the dominant conformation and the original conformation in the crystal structure of the complex protein was The binding mode is consistent with that of the original ligand. Docking lasaloxib under the same conditions revealed that lasaloxib fully occupies the original ligand binding pocket, with the carboxyl group of lasaloxib forming a strong interaction with TKT ARG401. The piperidine ring and substituted ethyl group form hydrophobic interactions with LEU125, HIS77, and HIS258. The piperidine ring oxygen atom forms a hydrogen bond with GLN189.
[0032] 2. Molecular dynamics simulation of lasaloxi and TKT transketolase
[0033] The best docking results of lasaloxi and TKT binding were simulated for 20 ns using the method reported in the literature (Cao, JF et al. Frontiers in physiology, 2022, 13, 990469). The trajectory analysis calculated the time curve of the root mean square deviation (RMSD) value over time, and the root mean square perturbation (RMSF) of the skeleton was calculated to evaluate the overall stability of the binding of lasaloxi and TKT. RMSF can represent the degree of freedom of each atom in the molecule. Figure 1 C shows that the RMSF values of the key residues of TKT do not fluctuate significantly, indicating that the binding of lasaloxib to TKT is stable. Figure 1 E is the RMSD-time curve, which shows that the RMSD fluctuation range of the system is approximately The simulation trajectory remained largely consistent, without significant distortion, indicating that lasaloxib can stably bind to the active pocket of the TKT receptor. Furthermore, conformational analysis based on full-time molecular dynamics simulations revealed that key amino acids were stable within 20 nanoseconds, further demonstrating a stable binding mode for lasaloxib. Therefore, based on molecular docking and dynamics simulation results, lasaloxib may act on TKT and play a regulatory role.
[0034] Example 2: Effect of Lasalox on the Proliferation of Hepatocellular Carcinoma Cells (CCK8 Assay)
[0035] The proliferation inhibition rate of lasaloxilide (sodium) in liver cancer cell lines HepG2, MHCC97H and HCCLM3 was calculated by 1X10 4 Cells were seeded in 96-well plates at 400 μM per well. After 24 hours of culture, cells were treated with 0.032, 0.16, 0.8, 4, 20, and 100 μM sorafenib and lasaloxicam (sodium). A drug-free blank control group was also established. Each group received 200 μL of culture medium, and triplicate wells were set up. After 24 hours of culture, 10 μL of CCK8 reagent was added to each well. The cells were incubated in an incubator for 30 minutes, and the optical density (OD) at a wavelength of 450 nm was measured with a microplate reader. Cell proliferation and survival rates and IC50 values were calculated.
[0036] Example 3: Effect of Lasalox on the Proliferation of Hepatocellular Carcinoma Cells (Clonogenic Assay)
[0037] The cells were cultured at 2×10 3Cells were seeded into 6-well plates at 100 μM and 20 μM lasalocid (sodium) per well. After 24 hours of culture, cells were treated with 10 and 20 μM lasalocid (sodium), respectively. A positive control group was treated with 20 and 40 μM sorafenib, respectively. The medium was replaced completely after 24 hours, and then every 4 days for a total of 12 days. The culture medium was discarded, and the cells were washed twice with PBS. The cells were then stained with 2% crystal violet for 10 minutes. The residual stain was rinsed with double-distilled water, and the cells were air-dried and photographed. The number of cell colonies formed was counted.
[0038] Example 4: Effect of Lasalox on the Migration of Hepatocellular Carcinoma Cells (Cell Scratch Test)
[0039] Cells were cultured at 1×10 6 Cells were inoculated into 6-well plates at 400 μg / well. After culturing for 24 h, the cell fusion status was observed. A 200 μL micropipette was used to evenly draw a line perpendicular to the middle of the 6-well plate. After the line was drawn, the culture medium was slowly aspirated with a pipette and the cells were washed once with culture medium to remove cell debris. The cells were divided into a control group, a sorafenib group, and a lasalocid (sodium) group, and 2.0 mL of culture medium was added to each group. The results were observed and photographed under an inverted microscope at 0 and 24 h, and the cell migration area was counted using Image software.
[0040] Example 5: Study on the toxicity of lasaloxib to normal cells
[0041] LO2 normal hepatocytes are counted as 1X10 4 Cells were seeded into 96-well plates at 100 μM per well. After 24 hours of culture, cells were treated with 3.125, 6.25, 12.5, 25, 50, and 100 μM sorafenib and lasaloxicam (sodium). A drug-free blank control group was also established. Each group received 200 μL of culture medium, with triplicate wells. After 24, 48, and 72 hours of culture, 10 μL of CCK8 reagent was added to each well. The cells were incubated in an incubator for 30 minutes, and the optical density (OD) at a wavelength of 450 nm was measured with a microplate reader. The cell proliferation inhibition rate and IC50 were calculated.
[0042] Example 6: Cellular thermal shift assay of lasalocib on TKT protein
[0043] Cellular thermal shift assay (CETSA) MHCC97H cells were treated with 0 or 40 μM lasalocid for 6 hours, washed with PBS, and lysed. Total protein from PBS-suspended cells supplemented with a protease inhibitor cocktail was divided into 12 equal portions, heated at 54, 58, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 82°C for 3 minutes, and frozen in liquid nitrogen for 3 minutes. Each sample underwent three freeze-thaw cycles. The corresponding loading buffer was added, and proteins were analyzed by western blotting.
[0044] Example 7: Tumor Inhibition Effect of Lasalox on Mice Transplanted with Hepatocellular Carcinoma Cells
[0045] All animal experiments were conducted in accordance with relevant national and local guidelines. Male BALB / c nude mice weighing 18–22 g were purchased from Beijing Laboratory Animal Co., Ltd. and housed in a pathogen-free environment (23 ± 2°C and 55 ± 5% humidity). After acclimation for 7 days, 5 × 10 6 MHCC97H cells. After 7 days, tumor size was determined using a micrometer caliper and the following formula: volume = (width 2 × length ÷ 2). Small rats with tumor volumes of 150-250 mm were randomly divided into 5 groups (5 rats / group): normal saline control group, positive control group (sorafenib 30 mg·kg -1 once daily), and lasalocid 15, 30, and 60 mg·kg -1 Treatment was administered intraperitoneally once daily for 8 days, and tumor size was measured every 2 days. Finally, mice were euthanized, and tumors were isolated and weighed.
[0046] Statistical analysis:
[0047] Statistical analysis was performed using GraphPad Prism 8.3.0 software. All experiments were performed in parallel at least three times, and data are presented as mean ± SEM. In all cases, statistical significance was determined at p < 0.05, *p < 0.05, and **p < 0.01.
[0048] Results of the docking between Lashaloxi and TKT
[0049] Figure 1 Discovery Studio software was used to dock lasaloxi with TKT (PDB: 6HAD), and a reasonable binding mode was selected for further molecular dynamics simulation. The results showed that the key amino acids HIS77, GLN189 and ARG401 of TKT all play a role in binding with lasaloxi. In the simulated solvent environment, 20ns of lasaloxi had good and stable binding with TKT. The binding mode of key amino acids remained basically unchanged, and the RMSF value of backbone amino acids was The RMSD value tends to be stable after running for 2ns.
[0050] Lasaloxib inhibits the proliferation of liver cancer cells (detected by CCK8 assay)
[0051] Figure 2Schematic diagram of cell survival rate after 24h treatment of three liver cancer cell lines, HepG2, MHCC97H, and HCCLM3, with different concentrations of lasaloxib and sorafenib. The results showed that lasaloxib inhibited the proliferation of liver cancer cells better than sorafenib in a concentration-dependent manner. The IC values of sorafenib in HepG2, MHCC97H, and HCCLM3 cells at 24h were 50 The values were 31.43, 28.48, and 40.38 μM, respectively, and the IC 50 The values are 5.34, 23.29, and 5.61 μM, respectively.
[0052] Lasaloxib inhibits the proliferation of liver cancer cells (clone formation assay)
[0053] Figure 3 Schematic diagram of the clone formation experiment to determine the proliferation of HepG2 cells treated with 5μM lasalocib and 20μM sorafenib, MHCC97H cells treated with 25μM lasalocib and 30μM sorafenib, and HCCLM3 cells treated with 5μM lasalocib and 20μM sorafenib for 24 hours. It can be seen from the figure that lasalocib has a slightly better effect on inhibiting the proliferation of liver cancer cells than sorafenib.
[0054] Lasaloxib inhibits the migration of liver cancer cells (cell scratch test)
[0055] Figure 4 Schematic diagram of the cell scratch assay to determine the proliferation of HepG2 cells treated with 10 μM lasalocib and 20 μM sorafenib, MHCC97H cells treated with 20 μM lasalocib and 40 μM sorafenib, and HCCLM3 cells treated with 10 μM lasalocib and 20 μM sorafenib for 24 hours. It can be seen from the figure that lasalocib has an inhibitory effect on liver cancer cell migration that is comparable to or even better than sorafenib.
[0056] Study on the cytotoxicity of lasaloxib to normal cells
[0057] Figure 5 Schematic diagram of CCK8 assay of cell survival rate of normal hepatocytes LO2 treated with different concentrations of lasaloxib and sorafenib at different times. As can be seen from the figure, the inhibitory effect of lasaloxib on hepatocyte proliferation is similar to that of sorafenib, and both are time-concentration dependent. The IC50 values of sorafenib at 24, 48, and 72 hours are 29.53, 10.20, and 12.23 μM, respectively, and the IC50 values of lasaloxib at 24, 48, and 72 hours are 24.74, 10.54, and 11.03 μM, respectively.
[0058] Cellular thermal shift assay of lasaloxib on TKT protein
[0059] Figure 6The cell heat shift method was used to verify whether lasalocid binds to the TKT protein. As shown in the figure, compared with the control, lasalocid changes the stability of the TKT protein after binding to the TKT protein.
[0060] Lasaloxib inhibits tumor growth in nude mice
[0061] Figure 7 control group, sorafenib 30 mg·kg -1 and lasalocid 15, 30, and 60 mg kg -1 Schematic diagram of the photos of tumor growth in the drug-treated group.
[0062] Figure 8 This is a schematic diagram of the effect of lasaloxib on the growth of MHCC97H nude mice xenograft tumors in different groups and time. It can be seen from the figure that the administration of 15 mg kg -1 , the 8-day treatment cycle had no significant effect on the body weight of tumor-bearing mice.
[0063] Figure 9 The effect of lasaloxib on the growth of MHCC97H nude mouse xenograft tumor is shown in the histogram of tumor weight in different groups of mice. As can be seen from the figure, the 8-day treatment cycle of 15 mg kg -1 Inhibits tumor growth in tumor-bearing mice.
[0064] Figure 10 The figure shows the effect of lasaloxib on the growth of MHCC97H nude mouse xenograft tumors, and the relationship between the tumor volume and time in different groups of mice. As can be seen from the figure, the 8-day treatment cycle of 15 mg kg -1 Inhibit tumor volume in tumor-bearing mice with 30mg·kg -1 Same as Sorafenib.
[0065] 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 or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.
Claims
1. The use of lasalocid as a transketolase inhibitor, characterized in that: Lasaloxib is used as a transketolase inhibitor. The lasaloxib is stably combined with the transketolase through molecular docking. Lasaloxib acts on the transketolase and plays a regulatory role.
2. The use of lasalocid as a transketolase inhibitor according to claim 1, characterized in that: The transketolase inhibitor is used to treat cancer.
3. The use of lasalocid as a transketolase inhibitor according to claim 2, characterized in that: The cancer is liver cancer, breast cancer, pancreatic cancer, lung cancer, melanoma, colon cancer, esophageal cancer, endometrial cancer, head and neck cancer, sarcoma, multiple myeloma, leukemia, urethral cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, cervical cancer, neuroendocrine tumor, prostate cancer, ovarian cancer, neuroblastoma.
4. The use of lasalocid as a transketolase inhibitor according to claim 3, characterized in that: Lasalox is active in vitro against liver cancer cells that highly express transketolase, and the experimental subjects are liver cancer cell-bearing mice.
5. The use of lasalocid as a transketolase inhibitor according to claim 4, characterized in that: The lasaloxib inhibits the activity of transketolase, thereby hindering the proliferation and migration of liver cancer cells and suppressing tumor growth.
6. The use of lasalocid as a transketolase inhibitor according to claim 1, characterized in that: The lasaloxib can be prepared into pharmaceutical preparations with conventional pharmaceutical excipients in pharmacy.
7. The use of lasalocid as a transketolase inhibitor according to claim 6, characterized in that: The pharmaceutical preparation is at least one of a capsule, a suspension, an emulsion, a solution, a syrup or an injection.
8. The use of lasalocid as a transketolase inhibitor according to claim 7, characterized in that: The pharmaceutical preparation can be administered orally or by injection.
9. The use of lasalocid as a transketolase inhibitor according to claim 8, characterized in that: The pharmaceutical preparation contains lasaloxib as the only active ingredient.