Application of ancitabine and analogues thereof in preparation of drugs for inhibiting cell pamosis
By using amecitabine and its analogs to inhibit the activity of PARP-1, the problem of failure to effectively inhibit cell Papara death in the prior art was solved, and effective inhibition of Papara death was achieved, providing a new direction for drug development.
Patent Information
- Application Number
- CN202510010033.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-03
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art has not disclosed the relationship between ancitabine and Palm death, and has not effectively inhibited Palm death in cell.
The activity of PARP-1 is inhibited by the use of amcitabine and its analogues, such as cytarabine or 5-fluorouracil, thereby blocking the occurrence of PARP-1.
Effective inhibition of cell Papa death has been achieved, new drug development directions, and important reference basis for the subsequent clinical application of amecitabine and its analogues.
Smart Images
Figure CN119868394A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug application, and specifically relates to the application of ancitabine and its analogs in the preparation of drugs for inhibiting cell death. Background Art
[0002] Parthanatos is a regulated cell death mode that depends on PARP-1 (poly ADP-ribose polymerase 1), also known as PARP-1-dependent cell death. In 2007, Ted Dawson and Valina Dawson of the Johns Hopkins University School of Medicine discovered this new form of regulated brain cell death. When cells are stressed and the genome is damaged, PARP-1 is overactivated, catalyzing the decomposition of intracellular nicotinamide adenine dinucleotide (NAD) into nicotinamide and poly ADP ribose (PAR). PAR accumulates in large quantities in the cell, and NAD is consumed in large quantities, inhibiting the activity of mitochondrial oxidative respiratory chain complex enzymes I to III, blocking the tricarboxylic acid cycle pathway, and impairing mitochondrial energy metabolism. Subsequently, AIF is released and carries macrophage migration inhibitory factor (MIF) to the nucleus, leading to chromatin condensation and large DNA fragmentation, ultimately mediating Parthanatos death.
[0003] Parkinson's disease is widely present in many diseases, especially in nervous system diseases, such as cerebral ischemia-reperfusion injury, Parkinson's disease, Alzheimer's disease, etc. Parkinson's disease is also involved in the pathogenesis of many diseases such as heart disease, retinal disease, gastrointestinal disease, lung disease, kidney disease, pancreatic disease, breast disease, etc.
[0004] Ancitabine is an anti-tumor drug that can interfere with multiple steps in the DNA replication process, including the regulation of DNA polymerase activity, the binding of topoisomerase IIa to double-stranded DNA, and other steps, thereby effectively inhibiting DNA synthesis. In addition to inhibiting DNA synthesis, ancitabine can also block the RNA transcription process. RNA is the transmitter of genetic information in cells, and blocking its transcription process can further inhibit the growth and division of tumor cells. Ancitabine treats diseases through multiple mechanisms such as inhibiting DNA synthesis, preventing RNA transcription, inducing apoptosis, enhancing immune system function, and antiviral effects.
[0005] However, during the search for ancitabine-related research, it was found that no existing technology has disclosed the relationship between ancitabine and Parkinson's disease death. Summary of the invention
[0006] The purpose of the present invention is to provide the use of ancitabine and its analogs in the preparation of drugs for inhibiting cell death (Parthanatos).
[0007] The technical solution adopted by the present invention is as follows:
[0008] The present invention provides the use of ancitabine in preparing a drug for inhibiting cell death.
[0009] The ancitabine has a blocking effect on Parkinson's disease death.
[0010] The present invention also provides the use of ancitabine analogs in preparing drugs for inhibiting cell death, wherein the ancitabine analogs are cytarabine or 5-fluorouracil.
[0011] The cytarabine or 5-fluorouracil has a blocking effect on Parkinson's death.
[0012] The present invention also provides a cell death inhibitor, which is ancitabine, cytarabine or 5-fluorouracil.
[0013] The present invention uses the classic cell model of MNNG-induced Parkinson's disease death in Hela cells to determine that ancitabine, cytarabine or 5-fluorouracil can inhibit Parkinson's disease death, and ancitabine has the strongest inhibitory effect.
[0014] The present invention determines that the blocking effect of ancitabine on PARP-1 is achieved by inhibiting the activity of PARP-1. Compared with the prior art, the present invention has the following beneficial effects:
[0015] The present invention discloses for the first time that ancitabine and its analogs cytarabine or 5-fluorouracil can inhibit cell death and can be used as cell death inhibitors. The blocking effect of ancitabine on cell death is achieved by inhibiting the activity of PARP-1. The technical solution provided by the present invention provides an important reference basis for the subsequent clinical application of ancitabine and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Schematic diagram of Hela cells pretreated with different concentrations of ancitabine (PI1) for 24 hours, then added with MNNG, and the cell death status after 12 hours;
[0017] Figure 2 The effect of different concentrations of ancitabine on cell viability was detected by CCK8;
[0018] Figure 3 To detect the inhibitory effect of different concentrations of ancitabine on MNNG-induced cell death for CCK8;
[0019] Figure 4 It is a two-dimensional diagram of the tolerance of ancitabine (Anc) to MNNG; the horizontal direction is the different concentrations of MNNG, and the vertical direction is the concentration gradient of Anc, the unit is μM; as the concentration of Anc increases, the tolerance to high concentration of MNNG increases;
[0020] Figure 5 This is a graph showing the inhibition of MNNG-induced cell death after cells were pretreated with ancitabine and its analogs for 10 hours;
[0021] Figure 6 This is a graph showing the inhibition of MNNG-induced cell death after 20-hour pretreatment of cells with ancitabine and its analogs;
[0022] Figure 7 The figure shows the effect of different concentrations of ancitabine on the expression of related proteins. Ancitabine does not affect the expression of PARP1 protein, but it significantly inhibits the activity of PARP1, which is manifested by a significant reduction in the PAR modified protein, the active product of PARP1.
[0023] Figure 8 After MNNG treatment, total cell protein was collected at different time periods and nuclear-cytoplasmic separation was performed to detect the changes in the content of protein AIF in different components; ANC inhibited the nuclear translocation of AIF protein.
[0024] Fig. 9 The inhibitory effect of ancitabine pretreatment duration on cell death; Pre-MNNG row corresponds to the ANC pretreatment duration before MNNG administration; Post-MNNG row corresponds to the administration of the specified drug after MNNG administration; OLA is the PARP1 inhibitor olaparib, PJ34 is another PARP1 inhibitor, and WT is the group without MNNG treatment;
[0025] Fig.10 Effect of ancitabine administration on PAR protein expression after MNNG. DETAILED DESCRIPTION
[0026] The present invention is described in detail below with reference to the embodiments.
[0027] Unless otherwise specified, the culture medium used in each embodiment is DMEM culture medium containing 10% by mass concentration of fetal bovine serum.
[0028] The solutions of each substance in each example were obtained by diluting each substance to a specified concentration using a DMEM culture medium containing 10% by mass concentration of fetal bovine serum.
[0029] The cultures involved in each embodiment were all cultured in a cell culture incubator at 37° C., 5% CO 2 in air and 100% humidity.
[0030] Example 1
[0031] Application of Ancitabine (ANC) in the preparation of drugs for inhibiting Parthanatos cell death
[0032] The classic cell model of Parthanatos induced by MNNG in Hela cells was selected. Hela cells were pretreated with DMSO, 30 μM PARP-1 inhibitor DPQ solution, 6 μM ancitabine solution, 12.5 μM ancitabine solution, 25 μM ancitabine solution, and 50 μM ancitabine solution for 24 hours (all referring to the final concentration dissolved in the cell culture medium), and then 60 μM MNNG solution was added for a further 15 min. The cells were washed three times with culture medium, and then 100 μL culture medium with the corresponding drug concentration was added for a further 24 h. The cell survival was observed under a microscope, as shown in FIG. Figure 1 As shown in the figure, it can be seen that the inhibitory effect of ancitabine is concentration-dependent, and at 12.5 μM, the cell viability is about 70% compared with the control group.
[0033] Hela cells were treated with 0, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, and 200μM ancitabine solutions for 24 hours, four groups were made for each concentration, and finally 10μL CCK8 solution was added to detect cell viability. The results are shown in Figure 2 As shown in the figure, it can be seen that ancitabine itself has a weak killing effect on Hela cells.
[0034] Hela cells were pretreated with DMSO, 30 μM PARP-1 inhibitor DPQ solution, 0.78125 μM ancitabine solution, 1.5625 μM ancitabine solution, 3.125 μM ancitabine solution, 6.25 μM ancitabine solution, 25 μM ancitabine solution, 50 μM ancitabine solution, 100 μM ancitabine solution, and 200 μM ancitabine solution for 24 hours, respectively, and then 60 μM NNG solution was added for further 15 min, the cells were washed three times with culture medium, and then 100 μL culture medium with corresponding drug concentration was added for further culturing for 24 hours. Finally, 10 μL CCK8 solution was added to detect cell viability. The results are shown in Figure 3 As shown, concentrations above 12.5 μM can effectively inhibit cell death.
[0035] Hela cells were treated with 0, 6.25μM, 12μM, 25μM, 50μM, 100μM, and 200μM ancitabine solutions for 24 hours, with four groups for each concentration. Then, 60μM, 80μM, 100M, and 200μM MNNG solutions were added for 15min, respectively. The cells were washed with culture medium for 3 times, and then 100μL culture medium of the corresponding drug concentration was added for 24h. Cell survival was observed under a microscope. Figure 4 As shown in the figure, it can be seen that the protective effect of ancitabine weakens with the increase of MNNG concentration.
[0036] Example 2
[0037] Ancitabine analogs inhibit MNNG-induced Parthanatos
[0038] After confirming that ancitabine has a significant inhibitory effect on Parthanatos in Hela cells, we further tested whether four structurally similar compounds of ancitabine also have the effect of blocking Parthanatos. These four compounds are cytarabine, capecitabine, 5-fluorouracil (5-FU), and gemcitabine.
[0039] Hela cells were pretreated with 25 μM, 50 μM, and 100 μM of the above substances for 10 hours and 20 hours, respectively, and then 60 μM MNNG solution was added for further 15 minutes. The cells were washed three times with culture medium, and then 100 μL of culture medium was added to each well for further 24 hours. The cell morphology was observed under a microscope. The results are as follows: Figure 5 and Figure 6 The results showed that among these four analogs, only Cytarabine and 5-FU had a lower inhibitory effect on cell death under MNNG toxicity damage, but the effects were not as good as Anc.
[0040] Example 3
[0041] Ancitabine prevents the occurrence of Parthanatos by inhibiting PARP-1 activity
[0042] In a six-well cell culture plate, Hela cells were pretreated with DMSO, 30 μM PARP-1 inhibitor olaparib (OLA) solution, 25 μM ancitabine solution, 50 μM ancitabine solution, and 100 μM ancitabine solution for 24 hours, and then 60 μM MNNG solution was added for 15 minutes. The cells were washed three times with culture medium, and then 1.5 mL of culture medium with the corresponding drug concentration was added for 30 minutes. The cells were lysed with lysis buffer, and the total amount of PAR-modified proteins was detected by protein electrophoresis and western blotting using PAR antibodies. The results are shown in FIG. Figure 7 As shown, according to the results of Western blotting, the effect of ancitabine in inhibiting PAR protein was comparable to that of the control group PARP inhibitor olaparib (OLA), and neither affected the expression level of PARP-1, which suggested that ancitabine may be a potential PARP1 inhibitor.
[0043] In a cell culture plate, Hela cells were pretreated with DMSO and 100 μM ancitabine solution for 24 hours, and then 60 μM MNNG solution was added for further treatment for 15 minutes. The cells were washed three times with culture medium, and then 10 mL of culture medium with the corresponding drug concentration was added for further culture for 6, 9, 12, and 15 hours. A group of Hela cells without any treatment (WT group) was added. The cells in each group were lysed with lysis solution and treated with the Biyuntian Nuclear and Cytoplasmic Isolation Kit to separate cytoplasmic and nuclear proteins. Protein electrophoresis and Western blotting were performed using AIF antibody for detection. The results are shown in Figure 8 As shown, the nuclear-cytoplasmic fractionation results showed that ancitabine could inhibit the nuclear translocation of apoptosis-inducing factor (AIF) induced by MNNG.
[0044] Fig. 9 It was shown that ancitabine pretreatment within 3 hours could not inhibit MNNG-induced cell death, which indicated that it takes a longer process for ancitabine to exert its inhibitory effect on MNNG cell death.
[0045] After MNNG administration, the designated drugs were added to the cell culture medium. Within 1 hour, the expression of PAR in the cell protein in the ancitabine group was not downregulated compared with the PARP inhibitor olaparib (OLA), e.g. Fig.10 This shows that although the effect of ancitabine in inhibiting cell death after 24 hours of pretreatment is comparable to that of the classic PARP inhibitor OLA, and it inhibits PARP1 activity, it cannot inhibit PARP1 activity when pretreated for 0.5-1 hour, indicating that its intrinsic mechanism of inhibiting PARP1 activity may be different from that of OLA, and it may indirectly inhibit PARP1 activity through the action of other proteins.
[0046] The detailed description of the use of ancitabine and its analogs in the preparation of drugs for inhibiting cell death by reference to the above examples is illustrative rather than restrictive, and several examples can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.
Claims
1. Application of ancitabine in the preparation of drugs for inhibiting cell death (Parthanatos).
2. The use according to claim 1, characterized in that: The ancitabine has a blocking effect on Parkinson's disease death.
3. The use of ancitabine analogs in the preparation of drugs for inhibiting cell death, characterized in that: The ancitabine analog is cytarabine or 5-fluorouracil.
4. The use according to claim 3, characterized in that: The cytarabine or 5-fluorouracil has a blocking effect on Parkinson's death.
5. A cell death inhibitor, characterized in that The cell death inhibitor is ancitabine.
6. A cell death inhibitor, characterized in that The cell death inhibitor is cytarabine.
7. A cell death inhibitor, characterized in that The cell death inhibitor is 5-fluorouracil.