Application of 7-hydroxy-4-methylcoumarin and analogue thereof in preparation of medicine for inhibiting Parthanatos cell death

Abstract

The invention discloses application of 7-hydroxy-4-methylcoumarin and an analogue thereof in preparation of a medicine for inhibiting Parthanatos cell death, and belongs to the technical field of medicine application. The 7-hydroxy-4-methylcoumarin and analogues of 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone or 6-hydroxy-4-methylcoumarin are disclosed for the first time, the 7-hydroxy-4-methylcoumarin and analogues of 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone or 6-hydroxy-4-methylcoumarin can inhibit Parthanatos cell death, the inhibitory effects of the 7-hydroxy-4-methylcoumarin and analogues of 8-formyl-4-methylumbelliferone and butyric acid-4-methylumbelliferone or 6-hydroxy-4- According to the technical scheme provided by the invention, an important reference basis is provided for subsequent clinical application of the 7-hydroxy-4-methylcoumarin and the analogues of the 7-hydroxy-4-methylcoumarin.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and specifically relates to application of 7-hydroxy-4-methylcoumarin and its analogs in preparing drugs for inhibiting Parthanatos 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, resulting in chromatin condensation and large DNA fragmentation, ultimately mediating Parthanatos.

[0003] Parthanatos is widely present in many diseases, especially playing a key role in nervous system diseases, such as cerebral ischemia-reperfusion injury, Parkinson's disease, Alzheimer's disease, etc. Parthanatos 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] 7-Hydroxy-4-methylcoumarin, also known as hymecromone, also known as 4-methylumbelliferone, is a heterocyclic aromatic compound. It is an important intermediate for synthetic fragrances, medicines, pesticides and dyes. At the same time, it also has high physiological activity and is widely used in pharmaceutical applications. As a choleretic, 7-hydroxy-4-methylcoumarin has a relaxing effect on the sphincter of the bile duct opening and has strong antispasmodic and analgesic effects. It can gently and continuously promote bile secretion, strengthen gallbladder contraction, and has a certain antibacterial effect. It has a significant choleretic effect, is conducive to the discharge of stones, and has a certain stone discharge effect on common bile duct stones. In addition, it can also be used to treat post-cholecystectomy syndrome. 7-Hydroxy-4-methylcoumarin is also an intermediate of the antiallergic drug sodium cromoglycate, providing an important raw material for the research and development and production of antiallergic drugs. 7-Hydroxy-4-methylcoumarin is considered to be the "core" molecule of biological insecticides, and its derivatives have a significant killing effect on mosquito eggs. 7-Hydroxy-4-methylcoumarin and its derivatives can also be used as detection reagents, such as for the rapid detection reagent methylumbelliferyl β-pyranogalactoside for synthesizing enzyme substrates. In addition, it also has the property of fluorescing and can be used as a fluorescent indicator and an acid-base indicator.

[0005] During the search for research related to 7-hydroxy-4-methylcoumarin, it was found that no prior art has disclosed the relationship between 7-hydroxy-4-methylcoumarin and Parthanatos. Summary of the invention

[0006] The object of the present invention is to provide the use of 7-hydroxy-4-methylcoumarin and its analogs in preparing drugs for inhibiting Parthanatos cell death.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The invention provides application of 7-hydroxy-4-methylcoumarin in preparing a drug for inhibiting Parthanatos cell death.

[0009] The 7-hydroxy-4-methylcoumarin has a blocking effect on Parthanatos.

[0010] The present invention also provides the use of 7-hydroxy-4-methylcoumarin analogs in preparing drugs for inhibiting Parthanatos cell death. The 7-hydroxy-4-methylcoumarin analogs are 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, or 6-hydroxy-4-methylcoumarin.

[0011] The 7-hydroxy-4-methylcoumarin analogue is 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, or 6-hydroxy-4-methylcoumarin, which has a blocking effect on Parthanatos.

[0012] The present invention also provides a Parthanatos cell death inhibitor, which is 7-hydroxy-4-methylcoumarin, 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, or 6-hydroxy-4-methylcoumarin.

[0013] The present invention uses the classic cell model of MNNG-induced Parthanatos in Hela cells to determine that 7-hydroxy-4-methylcoumarin, 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, and 6-hydroxy-4-methylcoumarin can inhibit Parthanatos cell death, and the inhibitory effects of the four are comparable.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention discloses for the first time that 7-hydroxy-4-methylcoumarin and its analogs 8-formyl-4-methylumbelliferone, butyrate-4-methylumbelliferone and 6-hydroxy-4-methylcoumarin can inhibit Parthanatos cell death and can be used as Parthanatos cell death inhibitors.

[0016] The technical solution provided by the present invention provides an important reference basis for the subsequent clinical application of 7-hydroxy-4-methylcoumarin and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The cell status diagram after Hela cells were treated with different concentrations of MNNG for 24 hours;

[0018] Figure 2 This is a statistical chart of cell viability detected by CCK8 after Hela was treated with different concentrations of MNNG;

[0019] Figure 3 This is a diagram of the cell status after Hela cells were pretreated with 20μM Olaparib, 20μM zVAD-fmk, 20μM Necrostatin-1, and 2mM 3MA for 24 hours, and then treated with 60μM MNNG for another 24 hours;

[0020] Figure 4This is a statistical chart of cell viability detected by CCK8 after Hela cells were pretreated with 20μM Olaparib, 20μM zVAD-fmk, 20μM Necrostatin-1, and 2mM 3MA for 24 hours, and then treated with 60μM MNNG for 15 minutes.

[0021] Figure 5 The chemical structure of 7-hydroxy-4-methylcoumarin (4-Methylumbelliferone, 4MU);

[0022] Figure 6 Hela cells were treated with 50, 100, 200, 400, 600, 800, and 1000 μM 4MU for 24 h, and cell viability was detected using CCK8;

[0023] Figure 7 Microscopic pictures of Hela cells pretreated with gradient concentrations (0-200 μM) of 4MU for 24 hours, treated with 60 μM MNNG for 15 minutes and then removed, and then cultured for another 24 hours;

[0024] Figure 8 CCK-8 was used to detect the effects of different concentrations of 4MU on the viability of Hela cells after 24 h of MNNG (60 μM, 15 min);

[0025] Fig. 9 The EC50 of 4MU was determined by concentration-cell viability curve fitting = 14.46 μM;

[0026] Fig.10 The micrographs are for MNNG (60 μM, 15 min) treatment and 0 μM, 50 μM, 100 μM, and 200 μM 4MU treatment 24 h later;

[0027] Fig.11 For MNNG (60 μM, 15 min) treatment, 0 μM, 50 μM, 100 μM, and 200 μM 4MU were added and treated for 24 h, and CCK8 was used to detect cell viability;

[0028] Fig.12 Figures AH in the figure are the chemical structures of 8 4MU analogs: coumarin (A); 7-hydroxycoumarin (B); 6-methylcoumarin (C); 6-hydroxy-4-methylcoumarin (D); 4μ8C (E); 4-MUB (F); 7-amino-4-methylcoumarin (G); 7-hydroxy-4-methylcoumarin-3-acetic acid (H); Figure IJ is a cell micrograph and CCK8 detection result after Hela cells were pretreated with 50μM concentration of 4MU and its 8 analogs for 24 hours and treated with MNNG (60μM, 15 minutes) for 24 hours. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the embodiments.

[0030] Unless otherwise specified, the culture medium used in each embodiment is DMEM culture medium containing 10% by mass concentration of fetal bovine serum.

[0031] 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.

[0032] 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.

[0033] Example 1

[0034] Application of 7-hydroxy-4-methylcoumarin in the preparation of drugs for inhibiting Parthanatos cell death

[0035] A classic cell model of Parthanatos induced by MNNG in Hela cells was selected. 12,000 Hela cells were seeded into a 96-well plate per well, 100 μL of culture medium was added to each well, and 70-80% density Hela cells were pretreated with DMSO, 30 μM MNNG solution, 40 μM MNNG solution, 50 μM MNNG solution, 60 μM MNNG solution, and 70 μM MNNG solution for 15 min, respectively. The cells were then washed three times with culture medium, and then 100 μL of culture medium was added to continue culturing for 24 h. The cell morphology was observed under a microscope, as shown in FIG. Figure 1 As described above. After adding 10 μL of CCK8 solution and culturing for 2 hours, the absorbance at 450 nm was measured using an ELISA instrument to detect cell viability. The cell viability statistics are shown in the figure below. Figure 2 As shown in the figure, it can be determined that the subsequent MNNG concentration is 60 μM, at which time the cell viability drops to about 7% of the control group.

[0036] After determining the concentration of MNNG, the Hela cells were pretreated with 20 μM PARP inhibitor Olaparib, 20 μM apoptosis inhibitor zVAD solution, 20 μM necrosis inhibitor Necrostatin-1 solution, and 2 mM autophagy inhibitor 3MA solution for 24 hours, respectively, using DMSO-treated Hela cell line as a control, and then added with 60 μM MNNG solution for 15 minutes. The cells were washed three times with culture medium, and then 100 μL culture medium was added for 24 hours. The cell morphology was observed under a microscope, as shown in FIG. Figure 3 As shown, 10 μL CCK8 solution was added to detect cell viability. The cell viability statistics are shown in Figure 4 As shown in the figure, only the PARP inhibitor Olaparib can inhibit MNNG-induced cell death. This shows that the cell death model constructed by treating Hela cells with MNNG is PARthanatos, which can be used as a cell model for studying Parthanatos in the future.

[0037] After determining the screening model, a compound library containing a variety of marketed drugs was constructed, and several drugs with the highest cell viability after treatment were selected for further rescreening based on the screening results. Specifically, Hela cells were pretreated with 60μM drug solution for 24 hours, then 60μM 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.

[0038] Since the single-hole experiment has errors, in order to enhance the reliability of the results, the initially screened drugs were rescreened for three rounds, and it was finally determined that 7-hydroxy-4-methylcoumarin has a protective effect on MNNG damage.

[0039] The protective effect of the screened 7-hydroxy-4-methylcoumarin was verified. Hela cells were pretreated with 50, 100, 200, 400, 600, 800, and 1000 μM 7-hydroxy-4-methylcoumarin (4MU) solutions for 24 hours, and then 60 μM MNNG solution was added for further treatment for 15 minutes. The cells were washed with culture medium for 3 times, and then 100 μL culture medium was added for further culture for 24 hours. Finally, 10 μL CCK8 solution was added to detect cell viability. The results are as follows: Figure 6 As shown, 7-hydroxy-4-methylcoumarin was found to have a slight toxic effect on cultured cells at concentrations greater than 200 μM.

[0040] Then, Hela cells were pretreated with 4MU at gradient concentrations of 1.5625μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, and 200μM for 24 hours, and then 60μM MNNG solution was added to continue treatment for 15min, washed with culture medium three times, and then 100μL culture medium was added to continue culture for 24h. It was found that 4MU had a significant blocking effect on Parthanatos, and within the gradient range, with the increase of 4MU concentration, the number of adherent cells and cell viability increased significantly, as shown in Figure 7 and Figure 8 As shown. The EC50 was determined to be 14.46 μM by fitting the 4MU concentration-cell viability curve. Fig. 9 shown.

[0041] Hela cells were directly treated with 60 μM MNNG solution for 15 minutes, and then different concentrations of 4MU were added. Cell survival was observed after 24 hours. The results showed that 4MU also had a protective effect on Hela cells damaged by MNNG without pretreatment, and the protective effect gradually increased with the increase of concentration. Fig.10 and Fig.11 shown.

[0042] Example 2

[0043] 7-Hydroxy-4-methylcoumarin analogs inhibit MNNG-induced Parthanatos

[0044] After confirming that 7-hydroxy-4-methylcoumarin has a significant inhibitory effect on Parthanatos in Hela cells, we further tested whether eight structurally similar compounds of 7-hydroxy-4-methylcoumarin also have the effect of blocking Parthanatos. Their structural formulas are as follows: Fig.12 As shown in Figure A. The eight compounds are coumarin, 7-hydroxycoumarin, 6-methylcoumarin, 6-hydroxy-4-methylcoumarin, 8-formyl-4-methylumbelliferone (4μ8C), 4-MUB (4-Methylumbelliferyi butyrate); 7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin-3-acetic acid.

[0045] Hela cells were pretreated with 50 μM of each of the above substances for 24 hours, and then 60 μM MNNNG solution was added for further treatment for 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 culture for 24 hours. The cell morphology was observed under a microscope. The results are as follows: Fig.12 As shown in Figure I, 10 μL of CCK8 solution was added and cultured for 2 h. The absorbance at 450 nm was measured to count the cell viability. The results are as follows Fig.12 As shown in Figure J, the results show that among these eight analogs, only 4μ8C, 4-MUB, and 6-hydroxy-4-methylcoumarin have similar effects on inhibiting MNNG toxicity as 4MU under MNNG toxicity damage. This shows that the basic structure of 7-hydroxy-4-methylcoumarin should be the main basis for its inhibition of cellular PARthanatos.

[0046] The detailed description of the use of 7-hydroxy-4-methylcoumarin and its analogs in the preparation of drugs for inhibiting Parthanatos cell death with 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 7-hydroxy-4-methylcoumarin in the preparation of drugs for inhibiting Parthanatos cell death.

2. The use according to claim 1, characterized in that: The 7-hydroxy-4-methylcoumarin has a blocking effect on Parthanatos.

3. Use of 7-hydroxy-4-methylcoumarin analogs in the preparation of drugs for inhibiting Parthanatos cell death, characterized in that: The 7-hydroxy-4-methylcoumarin analog is 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, or 6-hydroxy-4-methylcoumarin.

4. The use according to claim 3, characterized in that: The 7-hydroxy-4-methylcoumarin analogue is 8-formyl-4-methylumbelliferone, butyric acid-4-methylumbelliferone, or 6-hydroxy-4-methylcoumarin, which has a blocking effect on Parthanatos.

5. A Parthanatos cell death inhibitor, characterized in that The Parthanatos cell death inhibitor is 7-hydroxy-4-methylcoumarin.

6. A Parthanatos cell death inhibitor, characterized in that The Parthanatos cell death inhibitor is 8-formyl-4-methylumbelliferone.

7. A Parthanatos cell death inhibitor, characterized in that The Parthanatos cell death inhibitor is butyrate-4-methylumbelliferyl.

8. A Parthanatos cell death inhibitor, characterized in that The Parthanatos cell death inhibitor is 6-hydroxy-4-methylcoumarin.

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