Application of ubenimex in preparation of medicine for inhibiting Parthanatos cell death

Abstract

The invention discloses an application of ubenimex in preparation of a medicine for inhibiting Parthanatos cell death, the invention discloses that ubenimex can inhibit Parthanatos cell death and can treat Parthanatos by inhibiting Parthanatos cell death for the first time, and the blocking effect of ubenimex on Parthanatos is not realized by inhibiting PARP-1 activity, so that the application of ubenimex in preparation of the medicine for inhibiting Parthanatos cell death can be used for treating Parthanatos. The technical scheme provided by the invention provides an important reference basis for subsequent clinical application of ubenimex.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and particularly relates to application of ubenimex in preparing a drug for inhibiting Parthanatos cell death. Background Art

[0002] When PARP-1 in cells is in an overactivated state, it will cause the cells to die in a new way. In this process, PAR polymers are both cell death signals and mediators, so this mode of death is also called Parthanatos. Parthanatos is a new form of programmed cell death discovered in 2007.

[0003] The death mechanism of Parthanatos includes DNA damage, overactivation of PARP-1, depletion of NAD+ and ATP, nuclear translocation of AIF and MIF complex, and DNA fragmentation. For example, ionizing radiation, exposure to n-methyl-d-aspartate (NMDA) or DNA alkylating agent n-methyl-n0-nitro-n-nitrosoguanidine (MNNG), oxygen glucose deprivation (OGD) or long-term treatment with hydrogen peroxide and other reactive oxygen species (ROS) can directly induce DNA damage, leading to overactivation of PARP-1 and the production of long-chain and branched PAR polymers. The formation of PAR consumes intracellular NAD+ as a reaction substrate, and NAD+ resynthesis requires about 2-4 ATP molecules, further consuming endogenous functional molecules. PAR generated by PARP-1 overactivation is released from the nucleus to the cytoplasm, where it binds to apoptosis-inducing factor (AIF), resulting in the release of AIF from mitochondria. Once AIF is released from the outer surface of mitochondria into the cytoplasm, it binds to macrophage migration inhibitory factor (MIF) and transfers to the nucleus together. MIF is a DNA nuclease that cleaves genomic DNA into large fragments (approximately 20-50 kb fragments), leading to chromatin and nuclear condensation fragmentation, which in turn triggers cell death.

[0004] PARP1 is the first protein discovered to have PAR (poly ADP ribose) modification activity. It can synthesize linear and branched PAR polymers using NAD+ as a substrate, and modify some nuclear proteins and itself. PARP1 has dual functions: one is the protective effect. PARP1 can recruit some DNA repair-related proteins through PAR modification, and promote cell survival by playing a key role in DNA base excision repair, single-strand break repair and alternative non-homologous end joining to prevent the accumulation of potentially lethal DNA double-strand breaks. The other is the aforementioned cell death-promoting effect, that is, PARP1 promotes DNA breakage by promoting the translocation of AIF from mitochondria into the nucleus and activating the nuclease activity of MIF, which then triggers cell death, thereby eliminating defective cells that are considered unsalvageable to maintain the health of the entire body.

[0005] Previous studies have attempted to use PARP1 inhibitors to protect damaged neurons. PARP1 inhibitors do have the effect of maintaining the survival of nerve cells during use. However, since the activity of PARP1 in recruiting DNA repair proteins is also inhibited, damaged DNA cannot be properly repaired. Therefore, once the PARP1 inhibitor is removed, cells that have accumulated a large amount of DNA damage will continue to undergo Parthanatos. This also limits the use of PARP1 inhibitors in clinical applications as neuroprotective drugs. Therefore, there is an urgent need to develop new drugs that protect nerve cells by inhibiting the function of PARP1 downstream proteins. Blockers targeting Parthanatos are of great application value in the treatment of related diseases. However, there is still a large gap in the development of related drugs.

[0006] Ubenimex is traded under the name Bestatin, and its chemical name is N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl]-L-leucine. It is an organic compound with a chemical formula of C16H24N2O4. It is an inhibitor of cell surface aminopeptidase N / CD13 (APN / CD13) and aminopeptidase B. It mainly exerts anti-tumor effects by regulating the body's immune function and inducing tumor cell apoptosis. It is a white crystalline powder, easily soluble in glacial acetic acid, slightly soluble in water or methanol, and has a structure like Figure 1 As shown in Figure 1, it was officially launched in Japan in 1987 as an anticancer adjuvant drug with immunomodulatory function. Existing studies have shown that the main inhibitory target of ubenimex is aminopeptidase, which degrades the extracellular matrix and basement membrane by hydrolyzing the amino acids at the amino terminus of the peptide segment, thereby promoting tumor metastasis. Its expression is upregulated in some highly invasive and rapidly proliferating malignant tumors

[11] . 2+The hydrophobic active center inhibits the activity of aminopeptidases

[12] . Among the dozen aminopeptidases currently inhibited by ubenimex, aminopeptidase N (also known as CD13) and aminopeptidase B are the most studied, among which CD13 is the most important. In addition to the co-hydrolysis of the above aminopeptidases affecting tumor invasion and metastasis, CD13 is also related to angiogenesis, viral receptors, antigen presentation, and cytokine regulation

[13] . The high-efficiency probe with ubenimex activity developed by Harbut et al.

[14] in 2008 can more widely detect the functional characteristics of metalloaminopeptidases in different biological systems. In addition to targeting aminopeptidases, another inhibitory target of ubenimex is leukotriene A4 (LTA4) hydrolase, and Glu-296 is an important structural determinant in the binding process between the two

[12] . In addition, some researchers have found that ubenimex is also a competitive inhibitor of tumor dipeptidase. This enzyme was isolated and purified from mouse ascites tumor cells. Unlike the binding mode of aminopeptidase, the binding process of ubenimex to dipeptidase is slow but not tight

[15] . In summary, the targets of ubenimex are mainly aminopeptidase and LTA4 hydrolase. In the early days, it was believed that the anti-tumor activity of ubenimex in vivo kills tumor cells by affecting the body's immune response, such as activating immune cell macrophages, degrading and inactivating immune active factors, activating granulocytes and NK cells, and promoting anti-tumor nonspecific immune response. Subsequently, a large number of studies have found that even without immune response, ubenimex still has anti-tumor effects. Therefore, the anti-tumor effects of ubenimex include direct and indirect anti-tumor effects produced by immune regulation. In 2000, Imamura and Kimura found that the proliferation of human promyelocytic acute leukemia cell line HL60 cells could be inhibited by ubenimex. Ubenimex can also directly induce apoptosis in chronic myeloid leukemia K562 cells and inhibit cell proliferation. This process depends on the activation of caspase-3 and is also related to the phosphorylation of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3β (GSK-3β) and the inhibition of cyclin D1 expression. In normal colorectal cancer cells, the death associated protein kinase (DAPK) gene promoter is methylated, but after treating cells with ubenimex, the gene promoter is reversed to a demethylated state, and both DAPK mRNA and protein are restored to expression, cell proliferation is inhibited and apoptosis occurs. Other literature shows that ubenimex and its analogs bind to the BIR3 domain, causing the apoptosis inhibitor 1 to be automatically ubiquitinated and degraded, ultimately leading to apoptosis of tumor cells.For a long time, Ubenimex has been mainly used in the adjuvant treatment of tumors, improving the body's immunity, reducing adverse reactions in chemotherapy, and improving the quality of life of patients.

[0007] A large number of literatures show that Ubenimex is used in clinical trials and treatments of malignant tumors such as gastric cancer, ovarian cancer, colon cancer, lung cancer, acute myeloid leukemia, and kidney cancer. Ubenimex can improve the body's immune function through biological effects such as increasing the number of CD3+ and CD4+ lymphocytes in the patient's peripheral blood, improving the CD4+ / CD8+ ratio, improving NK cell activity, regulating immune factors, enhancing NO secretion ability and macrophage phagocytic function, and restoring peripheral blood myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) to normal levels.

[0008] During the search for relevant research on Ubenimex, it was found that there is currently no prior art that discloses the relationship between Ubenimex and Parthanatos, nor is there any prior art that discloses that Ubenimex treats related diseases by inhibiting Parthanatos cell death. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the use of ubenimex in preparing a drug for inhibiting Parthanatos cell death.

[0010] The present invention also provides the use of ubenimex in preparing a drug for treating Parkinson's disease. Ubenimex treats Parkinson's disease by inhibiting the death of Parthanatos cells.

[0011] The technical solution adopted by the present invention is as follows:

[0012] Use of ubenimex in the preparation of drugs for inhibiting Parthanatos cell death.

[0013] Furthermore, the ubenimex is ubenimex acetate or ubenimex hydrochloride.

[0014] The present invention uses the classic cell model of Parthanatos induced by MNNG in Hela cells to determine that ubenimex can inhibit the death of Parthanatos cells.

[0015] The present invention has determined that the blocking effect of Ubenimex on Parthanatos is not achieved by inhibiting the activity of PARP-1.

[0016] The present invention determines that ubenimex treats Parkinson's disease by inhibiting the death of Parthanatos cells. Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discloses for the first time that ubenimex can inhibit the death of Parthanatos cells.

[0018] The technical solution provided by the present invention provides an important reference basis for the subsequent clinical application of ubenimex. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure A in the middle is a statistical chart of the top 10 drugs with the highest cell viability in 24-hour CCK8 detection after 24-hour pretreatment with 50 μM MNNG for 15 minutes; Figure B is the chemical structure of Ubenimex;

[0020] Figure 2 Figure A in the middle shows the cell status of Hela cells after being pretreated with 30μM DPQ and 50μM Bestatin for 24 hours, and then treated with 50μM MNNG for 24 hours; Figure B shows the cell viability statistics of CCK8 detection after Hela cells were pretreated with 30μM DPQ and 50μM Bestatin for 24 hours, and then treated with 50μM MNNG for 15 minutes;

[0021] Figure 3 Figure A shows the cell diagram of cells treated with 50 μM MNNG for 15 minutes and 24 hours after the cells were treated with 0.78125 μM to 100 mM Ubenimex for 24 hours at two-fold increments; Figure B shows the cell viability statistics of CCK8 detection 24 hours after the cells were treated with 50 μM MNNG for 15 minutes and 24 hours after the cells were treated with 0.78125 μM to 100 mM Ubenimex for 24 hours at two-fold increments; Figure C shows the conversion of concentration-cell viability data into an s-shaped curve for determining the EC50 value of Ubenimex, and the calculated EC50 is 5.42 μM;

[0022] Figure 4 The results are shown in Figure 2, in which cells were pretreated with 50 μM ubenimex for 24 hours and then treated with 50 μM MNNG for 15 minutes. Proteins were extracted 20 minutes after treatment and PAR was detected by immunoblotting.

[0023] Figure 5 The fluorescence staining results of the slices in Example 3;

[0024] Figure 6 These are the results of fluorescent staining of slices at different ubenimex concentrations in Example 3. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the embodiments.

[0026] Unless otherwise specified, the culture medium used in each embodiment is DMEM culture medium containing 10% by mass concentration of fetal bovine serum.

[0027] 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.

[0028] 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.

[0029] Example 1

[0030] Drug screening identified ubenimex as an inhibitor of cell death in Parthanatos cells

[0031] The classic cell model of Parthanatos induced by MNNG in Hela cells was selected. 12,000 Hela cells were seeded into 96-well plates per well, and 100 μL of culture medium was added to each well. DMSO, 10 μM MNNG solution, 20 μM MNNG solution, 30 μM MNNG solution, 40 μM MNNG solution, and 50 μM MNNG solution were used to pretreat 70-80% density Hela cells for 15 min, and then washed with culture medium for 3 times, and then 100 μL of culture medium was added to continue culturing for 24 h, and the cell morphology was observed under a microscope. After adding 10 μL of CCK8 solution and culturing for 2 hours, the absorbance at 450 nm was detected by an enzyme reader to detect cell viability, and the subsequent MNNG concentration was determined to be 50 μM.

[0032] After determining the screening model, a compound library containing 279 marketed drugs was constructed. Based on the screening results, 12 drugs with the highest cell viability after treatment were selected for further rescreening. Specifically, Hela cells were pretreated with 50μM drug solution for 24 hours, then 50μM MNNG solution was added for further treatment for 15 minutes, washed with culture medium for 3 times, then 100μL culture medium was added for further culture for 24 hours, and finally 10μL CCK8 solution was added to detect cell viability. The cell viability statistics of the 12 drugs screened are shown in the figure below. Figure 1 As shown in A.

[0033] Since the single-hole experiment has errors, in order to enhance the reliability of the results, three rounds of rescreening were conducted on the 12 drugs initially screened, and it was finally determined that Ubenimex had a significant protective effect on MNNG damage.

[0034] The present invention mainly verifies the protective effect of a screened drug, Ubenimex, whose structural formula is as follows: Figure 1 As shown in Figure B. Hela cells were pretreated with 50 μM ubenimex solution for 24 hours, and the cell morphology was observed under a microscope. Figure 2 As shown in the upper row of Figure A in the figure, 50 μM MNNG solution was then added to continue treatment for 15 min, the cells were washed three times with culture medium, and then 100 μL of culture medium was added to continue culture for 24 h. The cell morphology was observed under a microscope. The results are shown in Figure 2 As shown in the lower row of Figure A, CCK8 was finally added to detect cell viability. The results are as follows Figure 2 As shown in Figure B. It can be seen that pretreatment with 50 μM ubenimex for 24 hours has a protective effect on MNNG-induced Parthanatos cell death. In order to further refine the effective concentration of ubenimex, Hela cells were pretreated with 0.7815 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM gradient concentrations of ubenimex solution for 24 hours, and then 50 μM MNNG solution was added for a further 15 minutes. The cells were washed three times with culture medium, and then 100 μL of culture medium was added for a further 24 hours. The cell morphology was observed under a microscope. The results are shown in Figure 2. Figure 3 As shown in Figure A, 10 μL of CCK8 solution was added to detect cell viability. The results are as follows Figure 3 As shown in Figure B, it was found that ubenimex protected cells from the toxic effects of MNNG in a concentration-dependent manner, with the protective effect reaching a peak at 25 μM and an EC50 of 5.42 μM. Figure 3 As shown in Figure C. When the concentration of ubenimex increased to 100 μM, the protective effect on cells decreased.

[0035] Example 2

[0036] Ubenimex does not prevent the occurrence of Parthanatos by inhibiting PARP-1

[0037] PARP-1 is the most widely studied ribozyme in the PARP protein family. PARP-1 functions as a DNA damage sensor in cells. After DNA damage, PARP-1 uses NAD+ as a substrate to catalyze the addition of single ADP-ribose or polymer PAR formed by ADP-ribose to different receptor proteins, including PARP-1 itself, which is the earliest response to DNA damage. Therefore, inhibiting PARP-1 can sensitize cancer cells to death by interfering with DNA repair and replication. PARP inhibitors have been approved by the FDA for the treatment of ovarian and breast cancer patients with homologous recombination (HR) defects. The upstream event of Parthanatos is the overactivation of PARP-1. Although PARP-1 inhibitors can significantly inhibit Parthanatos to avoid cell death and have become drugs, they are currently mainly used to inhibit the DNA repair function of PARP-1 to treat cancer. It is precisely because of the dual function of PARP-1 that while blocking Parthanatos by inhibiting PARP-1 to protect cells, it also affects the self-repair function of cells, so PARP-1 inhibitors have serious side effects in animal models of Parthanatos-related diseases.

[0038] In order to determine whether the blocking effect of ubenimex on Parthanatos is achieved by inhibiting PARP-1, the expression level of PAR, a product of PARP-1, was detected by Western blotting (using a PAR-modified antibody to detect the expression level of PAR, a product of PARP-1). 30 minutes after MNNG treatment was selected as the detection time. The results are as follows: Figure 4 As shown in the figure, pretreatment with 50 μM concentration of Ubenimex did not affect the production of PAR induced by MNNG, indicating that the blocking effect of Ubenimex on Parthanatos is not through inhibiting the activity of PARP-1.

[0039] Example 3

[0040] Application of ubenimex in preparing drugs for treating Parkinson's disease

[0041] Parkinson's disease (PD) is a neurodegenerative disease. The incidence rate in the population increases with the aging of the population. It is an age-related disease and a disabling disease. The treatment methods available today cannot significantly change the progression of the disease, causing a great socioeconomic burden. The clinical symptoms of PD mainly include movement disorders with bradykinesia, limb tremors, and muscle stiffness as the main manifestations, as well as non-movement disorders with sleep-wake cycle regulation disorders, cognitive disorders, mood and emotional disorders, autonomic dysfunction and pain as the main manifestations. Both aspects will increase with the development of the disease, seriously affecting the quality of life of patients. The present invention has found that ubenimex can treat Parkinson's disease by inhibiting the death of Parthanatos cells. The specific animal experiment process is as follows:

[0042] The mouse was placed in an inhalation anesthesia induction box, and the isopentane concentration was raised to 2% to induce anesthesia. After the mouse was fully anesthetized, the mouse head was connected to an anesthesia mask, and the isopentane concentration was adjusted to 1% to maintain anesthesia. The animal was fixed on a stereotaxic instrument (Jiangwan II type, China), and a 1.3 cm long incision was made along the sagittal line of the head to expose the anterior fontanelle. The needle insertion point was -1.5 mm beside the midline and +1.0 mm in front of the anterior fontanelle, and a small hole with a diameter of about 1 mm was made on the surface of the skull with a skull drill. The microinjector (Hamilton, 5 μL) slowly descended and injected the drug 3.5 mm below the skull at a speed of 0.5 μL / min. The needle was left in place for 5 minutes after each injection.

[0043] Normal control group: NS (injected into saline containing 1% ascorbic acid) was injected into the striatum stereotaxically for 14 days;

[0044] Model control group: 6-OHDA was injected into the striatum stereotaxically (2 μL of 5ug / ul6-OHDA solution containing 1% ascorbic acid) for 14 days;

[0045] Ubenimex intervention group: Ubenimex (2.5 mg / kg / d) was injected for 24 days, 6-OHDA was injected into the striatum stereotaxically on the 10th day, and 6-OHDA was injected for 14 days;

[0046] Mice were anesthetized with isoflurane and killed by transcardial perfusion with PBS. For immunofluorescence staining, brain tissue was perfused with 4% paraformaldehyde after PBS perfusion, fixed in 4% paraformaldehyde for 24 hours, and dehydrated in 30% sucrose solution for 3 days. Coronal sections of mouse brain were 20 μm thick and attached to anti-shedding slides, which were stored in a -80°C refrigerator for subsequent staining. Tissues of the striatum [caudate putamen (striatum), CPu] and substantia nigra (SN) regions on the modeling side and the healthy side were collected for Western blotting and RT-9PCR experiments. 60 to 70 sections were collected in the CPu and SN regions for immunofluorescence staining.

[0047] The results are shown in Figure 5. It can be seen from the figure that the number of green fluorescently labeled tyrosine hydroxylase (TH)-positive dopamine neurons on the injured side of the model control group injected with 6-OHDA decreased sharply, while the number of green fluorescently labeled TH-positive dopamine neurons in the ubenimex pretreatment group was basically the same as that in the normal control group. This shows that ubenimex pretreatment can protect dopaminergic neurons from Parthanatos cell death caused by 6-OHDA. Therefore, it is proved that ubenimex has the potential to treat Parkinson's disease by inhibiting Parthanatos cell death.

[0048] Exploration of Ubenimex Concentration

[0049] The 2.5 mg / kg / d ubenimex was diluted 2 times (A), 3 times (B), 4 times (C), and 5 times (D) respectively, and then the same experiment was carried out as the above ubenimex intervention group. The results are as follows Figure 6 As shown in the figure, it can be seen that 0.5 mg / kg / d ubenimex has no protective effect, which is manifested by a sharp decrease in the number of TH-positive dopamine neurons, similar to that of the model control group injected with 6-OHDA.

[0050] The detailed description of the use of ubenimex in the preparation of drugs for inhibiting Parthanatos cell death in the above reference examples is illustrative rather than restrictive, and several embodiments 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. Use of ubenimex in the preparation of drugs for inhibiting Parthanatos cell death.

2. The use of ubenimex in the preparation of a drug for inhibiting Parthanatos cell death according to claim 1, characterized in that: The ubenimex is ubenimex acetate or ubenimex hydrochloride.

3. The use of ubenimex in the preparation of a drug for treating Parkinson's disease, characterized in that: The ubenimex is used to treat Parkinson's disease by inhibiting the death of Parthanatos cells.

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