CN112159366A - Analog AclMDYPZ of NAT10 inhibitor Remodelin and application thereof - Google Patents
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- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention provides an analogue AclMDYPZ of a NAT10 inhibitor Remodelin and application thereof, relating to the technical field of biological medicine; with increasing doses of AC1MDYPZ, cell survival decreased gradually, in a dose-dependent relationship; the AC1MDYPZ can obviously increase the apoptosis rate and is in a dose-dependent relationship; the AC1MDYPZ can obviously increase the activity of Caspase-3, 9 and is in a dose-dependent relationship; the AC1MDYPZ can obviously increase BaxmRNA expression and reduce Bcl-2mRNA expression, and the Bax mRNA expression is gradually increased and the Bcl-2mRNA expression is gradually reduced along with the gradual increase of the dosage of the AC1MDYPZ in a dose-dependent relationship.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an analogue AclMDYPZ of a NAT10 inhibitor Remodellin and application thereof.
Background
Gastric cancer is an important health problem and is the fourth most common cancer and the second most cause of cancer death worldwide[1]. There are over 95 million diagnosed cases per year. Stomach cancer (GC) causes great pain to the mind and body of a patient and requires early treatment. Patients with early gastric cancer can be completely cured by an effective method[2]. But when advanced to the end stage, the recurrence rate of the treatment increases due to metastasis[3-4]. Although chemotherapy and radiotherapy have been used in the treatment to date, the recurrence rate due to metastasis is still extremely high and these procedures cause physiological and psychological distress to the patient[5]. Therefore, there is an urgent need to find a new therapeutic target to solve this problem.
N-acetyltransferase 10(NAT10, or hALP, a human N-acetyltransferase-like protein) is a nucleolin with lysine acetylation activity, including GNAT, RNA helicase and tRNA binding domain[6]。
Previous studies have shown that NAT10 has lysine acetylase activity and its substrates include histone, tubulin, tRNA and mRNA[6-8]. Therefore, NAT10 has been shown to be involved in a variety of cellular activities, such as ribosome biosynthesis, transcription and translation. High expression of NAT10 was found in cellular stress and various human cancers. The distribution of NAT10 and its role in cell division make this protein play an important role in cancer cell proliferation[9]. In colorectal cancer, NAT10 is regulated by glycogen synthase kinase-3 beta (GSK-3 beta), participates in Wnt signal pathway, and plays a key role in p53 activation through acetylation of p53[9-10]. In addition, NAT10 can regulate EMT of hepatocellular carcinoma and promote metastasis, and high expression of NAT10 is related to poor survival rate of patients[11-12]。
Meanwhile, the authors find that significant difference exists in the influence of high and low expression of NAT10 on the survival rate of clinical gastric cancer patients through database query (P ═ 4.4e-07), and the survival time of patients with high expression of NAT10 is obviously shortened.
Recent experiments prove that the inhibition of the function of the acetyltransferase protein NAT10 can reduce the migration and invasion of cancer cells[13-15]. Additional studies have shown that inhibition of NAT10 can attenuate doxorubicin resistance in breast cancer by reversing EMT. Remodelin is a small molecule compound that can specifically target and inhibit the N-acetyltransferase NAT10[16]. In addition, Remodelin can inhibit invasion and migration of gastric cancer cells under hypoxic conditions[17]. The NAT10 inhibitor remodelain can be a potential anticancer drug[18]。
Reference documents:
[1]Torre LA,Bray F,Siegel RL,etal.Global cancer statistics,2012.CA.2015,65(2):87–108.
[2]Bang CS,Baik GH,Shin IS,etal.Helicobacter pylori eradication for prevention ofmetachronous recurrence after endoscopic resection ofearly gastric cancer.J Korean Med Sci,2015,30(6):749–56.
[3]Riihimaki M,Hemminki A,Sundquist K,etal.Metastatic spread in patients with gastric cancer.Oncotarget,2016,7(32):52307–16.
[4]Bausys R,Bausys A,Vysniauskaite I,etal.Risk factors for lymph node metastasis in early gastric cancer patients:report from Eastern Europe country–Lithuania.BMC Surgery,2017,17(1):108.
[5]Karimi P,Islami F,Anandasabapathy S,etal.Gastric cancer:descriptive epidemiology,risk factors,screening,and prevention.Cancer Epidemiol Biomarkers Prev,2014,23(5):700-13.
[6]Zhang H,Hou W,Wang HL,etal.GSK-3beta-regulated N-acetyltransferase 10is involved in colorectal cancer invasion.Clin Cancer Res,2014,20(17):4717–4729.
[7]Lv J,Liu H,Wang Q,etal.Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation ofhTERT.Biochem Biophys Res Commun,2003,311(2):506–513.
[8]Chi YH,Haller K,Peloponese JM Jr,etal.Histone acetyltransferase hALP and nuclear membrane protein hsSUN1 function in de-condensation of mitotic chromosomes.J Biol Chem,2007,282(37):27447–27458.
[10]Liu X,Tan Y,Zhang C,etal.NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.EMBO Rep,2016,17:349-366.
[11]Zhang H,Hou W,Wang HL,etal.GSK-3beta-regulated N-acetyltransferase 10is involved in colorectal cancer invasion.Clin Cancer Res,2014,20:4717-4729.
[12]Ma R,Chen J,Jiang S,etal.Up regulation of NAT10 promotes metastasis of hepatocellular carcinoma cells through epithelial-to-mesenchymal transition.Am J Transl Res,2016,8:4215-4223.
[13]Zhang X,Liu J,Yan S,etal.High expression ofN-acetyltransferase 10：a novel independent prognostic marker of worse outcome in patients with hepatocellular carcinoma.Int J Clin Exp Pathol,2015,8:14765-14771.
[14]Balmus G,Larrieu D,Barros AC,etal.Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome.Nat Commun,2018,9(1):1700.
[15]Oh TI,Lee YM,Lim BO,etal.Inhibition of NAT10 suppresses melanogenesis and melanoma growth by attenuating microphthalmia-associated transcription factor(MITF)expression.Int J Mol Sci,2017,18(9):1924.
[16]Zhang X,Chen J,Jiang S,etal.N-acetyltransferase 10enhances doxorubicin resistance in human hepatocellular carcinoma cell lines by promoting the epithelial-tomesenchymal transition.Oxid Med Cell Longev,2019,7561879.
[17]Larrieu D,Britton S,Demir M,etal.Chemical inhibition of NAT10corrects defects oflaminopathic cells.Science,2014,344(6183):527–532.
[18]Wu J,Zhu H,Wu J,etal.Inhibition of N-acetyltransferase 10using remodelin attenuates doxorubicin resistance by reversing the epithelial-mesenchymal transition in breast cancer.Am J Transl Res,2018,10(1):256-264.Published 2018Jan 15.
[19]Larrieu D,Britton S,Demir M,etal.Chemical inhibition of NAT10corrects defects oflaminopathic cells.Science,2014,344:527-532.
disclosure of Invention
In view of the above, the present invention aims to provide an analog, AClMDYPZ, of a NAT10 inhibitor Remodelin and an application thereof, which can inhibit gastric cancer cell proliferation, reduce anti-apoptotic protein Bcl-2mRNA expression, increase the content of the pro-apoptotic protein Caspase-3, 9 and Bax mRNA expression, and have potential for treating gastric cancer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an analogue, AclMDYPZ, of a NAT10 inhibitor Remodelin, wherein the structural formula of the AclMDYPZ is shown as a formula I;
preferably, the Compound CID of the ACLMDYPZ is 2845182, and the chemical formula is C19H19N3S, molecular weight 321.442 g/mol.
The invention also provides application of the ACLMDYPZ in preparation of a medicine for inhibiting gastric cancer cell proliferation and promoting gastric cancer cell apoptosis.
The invention also provides application of the AClMDYPZ in preparing a medicine for treating gastric cancer.
The invention also provides a medicine for inhibiting the proliferation of gastric cancer cells and promoting the apoptosis of the gastric cancer cells, and the effective component of the medicine comprises the ACLMDYPZ.
The invention also provides a medicine for treating gastric cancer, and the effective component of the medicine comprises the AclMDYPZ.
Preferably, the medicine also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the active ingredients of the medicine can reduce the expression of anti-apoptotic protein Bcl-2mRNA, and increase the content of the pro-apoptotic protein Caspase-3, 9 and the expression of Bax mRNA.
The invention provides an analogue ACLMDYPZ of NAT10 inhibitor Remodelin, the chemical name of the analogue is 4- (4-methylpheny) -N- (1-phenylpropylideneamino) -1, 3-thiazole-2-amine) (Compound CID:2845182), the chemical formula is C19H19N3S, molecular weight 321.442 g/mol. According to the invention, through CCK8 experiment of in-vitro antitumor activity evaluation, the result shows that the cell survival rate is remarkably different from 4 mu mol/L (P) in the dose compared with the drug-free group<0.05). With increasing doses of AC1MDYPZ, cell survival declined gradually, in a dose-dependent relationship. The half inhibitory concentration IC50 value was 27.6. mu. mol/L at 48h of action. The apoptosis condition is further detected by a flow cytometer, and the result shows that the apoptosis rate is remarkably different from the dosage of 4 mu mol/L (P) compared with the group without the drug<0.05). The AC1MDYPZ can obviously increase the apoptosis rate. With the gradual increase of the dosage of the AC1MDYPZ, the apoptosis rate gradually increases, and is in a dose-dependent relationship. The ELISA detects the activity of the Caspase-3, 9 in the cells, and the result shows that the activity of the Caspase-3, 9 in the cells is remarkably different from that in a drug-free group from 4 mu mol/L (P)<0.05). AC1MDYPZ can obviously increase Caspase-3, 9 activity. Caspase-3, 9 activity was increased with increasing doses of AC1MDYPZ in a dose-dependent relationship. The expression of Bcl-2 and Bax mRNA in the cells is detected by qPCR, and the result shows that the expression of the Bcl-2mRNA and the Bax mRNA in the cells is remarkably different from that in a drug-free group from 4 mu mol/L (P)<0.05). AC1MDYPZ can obviously increase Bax mRNA expression and reduce Bcl-2mRNA expression. With the increasing dose of AC1MDYPZ, Bax mRNA expression gradually increased, and Bcl-2mRNA expression gradually decreased in a dose-dependent relationship.
Drawings
FIG. 1 is a graph showing the growth inhibitory effect of AclMDYPZ on human gastric cancer cell lines, in which the bar graph represents the average of three experiments and the error line represents the Standard Deviation (SD); denotes 0.01< P <0.05 for each group compared to the non-administered group; p <0.01 for each group compared to the non-administered group, the same applies below;
FIG. 2 is a graph showing the effect of ACLMDYPZ on intracellular Caspase-3 activity;
FIG. 3 is a graph showing the effect of ACLMDYPZ on intracellular Caspase-9 activity;
FIG. 4 shows the expression of intracellular Bcl-2mRNA by ACLMDYPZ;
FIG. 5 shows the expression of intracellular Bax mRNA by AclMDYPZ;
FIG. 6 is a graph of the effect of ACLMDYPZ on tumors in HepG2 tumor-bearing mice.
Detailed Description
The invention provides an analogue, AclMDYPZ, of a NAT10 inhibitor Remodelin, wherein the structural formula of the AclMDYPZ is shown as a formula I;
the Compound CID of the ACLMDYPZ is 2845182, and the chemical formula is C19H19N3S, molecular weight is 321.442g/mol, chemical name is 4- (4-methylpheny) -N- (1-phenylpropylideneamino) -1, 3-thiazole-2-amine). The ACLMDYPZ can inhibit the proliferation of gastric cancer cells, reduce the expression of anti-apoptosis protein Bcl-2mRNA, increase the content of apoptosis-promoting protein Caspase-3, 9 and the expression of Bax mRNA, and has the potential of treating gastric cancer.
The invention provides application of the ACLMDYPZ in preparation of a medicine for inhibiting gastric cancer cell proliferation and promoting gastric cancer cell apoptosis.
The invention provides application of the AclMDYPZ in preparing a medicine for treating gastric cancer.
The invention provides a medicine for inhibiting gastric cancer cell proliferation and promoting gastric cancer cell apoptosis, wherein the effective component of the medicine comprises the AclMDYPZ. The medicament of the invention preferably also comprises pharmaceutically acceptable auxiliary materials, and the dosage form of the medicament is not particularly limited, and different auxiliary materials can be used according to different dosage forms. The effective components of the medicine of the invention can preferably reduce the expression of anti-apoptotic protein Bcl-2mRNA, and increase the content of the pro-apoptotic protein Caspase-3, 9 and the expression of Bax mRNA.
The invention provides a medicine for treating gastric cancer, and the effective component of the medicine comprises the AclMDYPZ. The drugs of the present invention are preferably the same as those described above and will not be described herein.
The invention provides an AClMDYPZ analogue of the NAT10 inhibitor Remodelin and its use as described in detail in the examples below, which should not be construed as limiting the scope of the invention.
Example 1
CCK8 experiment is adopted to evaluate the growth inhibition effect of AC1MDYPZ on human gastric cancer cell strain, the cell survival rate and IC50And (4) carrying out measurement. Stomach cancer SGC7901 cells at 2X 104Individual cells/well were seeded into 96-well plates and allowed to adhere overnight. The following day 0, 2, 4, 8, 16, 32, 64. mu. mol/LAC1MDYPZ was added, and 10. mu.L of CCK8 solution (MCE, USA) was added to each well after 48 hours, and cultured at 37 ℃ for 4 hours. Absorbance was measured at 450nm using a microplate reader (Thermo full automatic MK3, Saimer Feishel USA). Cell viability and half inhibitory concentration IC50 values were calculated. Cell survival (%) × (drug-treated OD-blank OD)/(solvent-control OD-blank OD) × 100%.
As shown in FIG. 1, the cell viability was significantly different from that in the case of the drug-free group at the dose of 4. mu. mol/L (P)<0.05). With increasing doses of AC1MDYPZ, cell survival declined gradually, in a dose-dependent relationship. Half inhibitory concentration IC at 48h50The value was 27.6 umol/L.
Example 2
Apoptosis was detected following AC1MDYPZ action using flow cytometry. SGC7901 cells were seeded in 6-well plates containing 2mL of medium per well, and after 24 hours 0, 2, 4, 8, 16, 32, 64. mu. mol/LAC1MDYPZ was added, and after 48h incubation, cell suspensions were prepared after digesting each set of cells with 0.1% pancreatin (without EDTA). The cells were collected after centrifugation at 1000r/min for 5min, 5. mu.l of Annexin-V and 10. mu.l of PI dye were added, respectively, and mixed well and reacted in dark for 15min, and the cells were apoptotic by flow cytometry (Millipore Guava easy cell, Merck Mitigo, USA) within 1 h.
As shown in Table 1, the apoptosis rate was significantly different from the dose of 4. mu. mol/L (P <0.05) compared to the drug-free group. The AC1MDYPZ can obviously increase the apoptosis rate. With the gradual increase of the dosage of the AC1MDYPZ, the apoptosis rate gradually increases, and is in a dose-dependent relationship.
TABLE 1 Effect of AC1MDYPZ on the apoptosis Rate of SGC7901 cells
Note: p <0.05 compared to 0 umol/L; p <0.01 compared to 0 umol/L;
example 3
And (3) detecting the activity of Caspase-3, 9 in the cells. Caspase-3, 9 activity in cells is detected by applying a Caspase-3, 9 activity detection kit (Aimeijie Biotech, Inc.). After each group of cells was subjected to 0, 2, 4, 8, 16, 32, 64. mu. mol/LAC1MDYPZ for 48 hours, the supernatant culture solution was discarded, and the cells were trypsinized and collected into a cell culture solution for later use. 600g at 4 ℃, centrifugating for 5min, and collecting cells. The supernatant was removed, lysate was added, cells were lysed in an ice bath, and the cells were centrifuged at 16,000g at 4 ℃ for 12 min. The supernatant was collected, and the protein concentration was determined and adjusted to 2 mg/mL. Configuring a reaction system according to the requirements of the specification, detecting and adding Caspase-3 chromogenic substrate Ac-DEVD-pNA (2mmol/L) by Caspase-3, and detecting and adding Caspase-9 chromogenic substrate Ac-LEHD-pNA (2mmol/L) by Caspase-9. After incubation at 37 ℃ for 100min, absorbance at 405nm was measured using a microplate reader (Thermo-electric Thermo full-automatic MK3, siemer feishel, usa). The absorbance values of the normal groups were taken as 100% with the normal groups as a reference, and the relative Caspase-3, 9 activities of the groups were expressed by the measured optical density values of the experimental group/optical density values of the normal group, respectively.
The results are shown in FIGS. 2-3, and compared with the group without the drug, the activity of the Caspase-3, 9 in the cells is significantly different from the dose of 4 mu mol/L (P < 0.05). AC1MDYPZ can obviously increase Caspase-3, 9 activity. Caspase-3, 9 activity was increased with increasing doses of AC1MDYPZ in a dose-dependent relationship.
Example 4
And detecting the expression of apoptosis related genes Bcl-2 and Bax mRNA in the cells by real-time fluorescence quantitative PCR. Subjecting the cells to 0, 2, 4, 8, 16, 32, 64 μmol/LAC1MDYPZ treatment for 48 hr, extracting total RNA from the cells by Trizol method, and measuring OD with ultraviolet spectrophotometer260And OD280The absorbance, the concentration and purity of the RNA, OD260/OD280>1.8. PrimeScript Using reverse transcription kitTMRT reagent kit with gDNAeraser (Dalibao bioengineering Co., Ltd.) reverse transcribes the extracted total RNA into cDNA. Then, the cDNA after reverse transcription was used as a template, GAPDH was used as an internal control, and Green was usedTMPremix Ex TaqTMII (Tli RNaseH Plus) (Dalianbao bioengineering Co., Ltd.) the fluorescent quantitation kit was subjected to fluorescent quantitation PCR amplification on a Roche LightCycler96 fluorescent quantitation PCR instrument. Prepare 20 μ L reaction system: 2 μ L cDNA, 10 μ L TB GreenPremix Ex TaqII (2X), 0.8 μ L PCR Forward Primer (10 μ M), 0.8 μ L PCR Reverse Primer (10 μ M), 6.4 μ L sterile deionized water. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation 95 ℃ for 5s, annealing extension 60 ℃ for 20s, 35 cycles were repeated. The relative expression level of each gene is expressed as 2-. DELTA.Ct. The experiment was repeated 3 times in total. The primer sequences are as follows:
bax upstream (SEQ ID NO. 1): 5'-AGGATTGTGGCCTTCTTTGAG-3', respectively;
downstream (SEQ ID NO. 2): 5'-CCCACCGAACTCAAAGAAGG-3', respectively;
bcl-2 upstream (SEQ ID NO. 3): 5'-GGTTTCATCCAGGATCGAGCAGG-3', respectively;
downstream (SEQ ID NO. 4): 5'-ACAAAGATGGTCACGGTCTGCC-3', respectively;
GAPDH upstream (SEQ ID NO. 5): 5'-AGTGGGGTGATGCTGGTGCTG-3', respectively;
downstream (SEQ ID NO. 6): 5'-CGCCTGCTTCACCACCTTCTT-3' are provided.
The results are shown in FIGS. 4-5, where the expression of cellular Bcl-2mRNA and Bax mRNA was significantly different from the dose of 4. mu. mol/L (P <0.05) compared to the drug-free group. AC1MDYPZ can obviously increase BaxmRNA expression and reduce Bcl-2mRNA expression. With the increasing dose of AC1MDYPZ, Bax mRNA expression gradually increased, and Bcl-2mRNA expression gradually decreased in a dose-dependent relationship.
Example 5
Effect of AC1MDYPZ on tumor volume and tumor suppression rate in SGC7901 solid tumor mice. SGC7901 cells were cultured at 37 ℃ in 5% CO2And (5) in a constant-temperature incubator, carrying out passage once for 2-3 d. When the cells divide exponentially, they are digested with pancreatin and washed twice with PBS to give cells with a density of 1X 107Single cell suspension per ml. A1 ml syringe is used for injecting 0.2ml of cell suspension into a mouse (with the weight of 22-25 g, purchased from the center of experimental animals of Henan province (license number SCXK 2015-0005)) after the skin of the right axilla is injected for about 5mm, and about 7 days, the mouse has rice grain-like nodules and tough texture under the skin of the right axilla. After 2 weeks, the mice were sacrificed, the tumors were completely dissected and weighed. According to the formula: the tumor inhibition rate is calculated by taking the average tumor weight of the control mice-the average tumor weight of the experimental mice)/the average tumor weight of the control mice.
The results showed that the tumor weights were less in both the high and low dose groups of AClMDYPZ than in the model group 2 weeks after administration (fig. 6). The differences are all statistically significant (P < 0.05-0.01). Both the AClMDYPZ high and low dose groups had higher tumor inhibition rates, with the high dose group being higher than the lower dose group (table 2).
TABLE 2 influence of ACLMDYPZ on tumor weight and tumor suppression Rate in HepG2 tumor-bearing mice
(Note: P <0.05, P <0.01) compared to model group)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. An analog, AClMDYPZ, of the NAT10 inhibitor Remodelin, wherein the structural formula of the AClMDYPZ is shown in formula I;
2. the AClMDYPZ of claim 1, wherein the Compound CID of the AClMDYPZ is 2845182, and the chemical formula is C19H19N3S, molecular weight 321.442 g/mol.
3. The use of the AclMDYPZ of claim 1 or 2 in the preparation of a medicament for inhibiting gastric cancer cell proliferation and promoting gastric cancer cell apoptosis.
4. Use of the AClMDYPZ of claim 1 or 2 for the manufacture of a medicament for the treatment of gastric cancer.
5. A drug for inhibiting the proliferation and promoting the apoptosis of gastric cancer cells, wherein the active ingredient of the drug comprises the AclMDYPZ of claim 1 or 2.
6. A medicament for treating gastric cancer, which comprises the AClMDYPZ according to claim 1 or 2 as an active ingredient.
7. The medicament of claim 5 or 6, wherein the medicament further comprises pharmaceutically acceptable auxiliary materials.
8. The drug of claim 5 or 6, wherein the active ingredient of the drug is capable of reducing the expression of anti-apoptotic protein Bcl-2mRNA, increasing the content of pro-apoptotic protein Caspase-3, 9 and increasing the expression of Bax mRNA.
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