CN109942503B - Small molecule inhibitor AZIN18 and application thereof in pharmacy - Google Patents
Small molecule inhibitor AZIN18 and application thereof in pharmacy Download PDFInfo
- Publication number
- CN109942503B CN109942503B CN201910273003.5A CN201910273003A CN109942503B CN 109942503 B CN109942503 B CN 109942503B CN 201910273003 A CN201910273003 A CN 201910273003A CN 109942503 B CN109942503 B CN 109942503B
- Authority
- CN
- China
- Prior art keywords
- azin18
- cells
- small molecule
- molecule inhibitor
- polyamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention provides a small molecular inhibitor AZIN18, which has a structural formula as follows:
the invention relates to application of a small molecular inhibitor AZIN18 in preparing a medicament for inhibiting an enzyme-resistant inhibitory factor. The results show that: AZIN18 inhibits human prostate cancer PC3 cell proliferation, reduces ODC protein and polyamine content in cells, changes PC3 cell growth cycle, and induces PC3 cell to generate apoptosis.
Description
Technical Field
The invention provides a small molecule inhibitor for inhibiting an enzyme-resistant inhibitory factor, and simultaneously provides application of the small molecule inhibitor in preparing a medicament for treating tumor diseases.
Background
Polyamines are basic regulators of cell growth and development, and precise regulation of polyamine metabolism is essential for normal life activities. It has been found that dysregulation of polyamine metabolism is closely associated with the development and progression of a variety of diseases, including cancer, inflammation, atherosclerosis, stroke, renal failure and diabetes. The rapid cell division is highly dependent on the intracellular polyamine content, and thus the total polyamine content in tumor cells is also significantly higher than in normal cells. Research shows that the increase of the intracellular polyamine level can promote the growth and invasion and metastasis of tumors, and the decrease of the polyamine content can inhibit the proliferation of tumor cells, so that the regulation of polyamine metabolic pathways becomes an important means for preventing and treating tumors.
Under normal physiological conditions, the polyamine content in cells is regulated and controlled by complex synthesis, catabolism and transport mechanisms, and because the polyamine anabolism rate-limiting enzyme ODC plays the most important role in controlling the polyamine content in cells, the research on the regulation and control of the enzyme is most intensive. Anti-enzymes (AZs) are naturally occurring ODC inhibitors in cells that affect intracellular polyamine levels by a dual effect: (1) binds to ODCs to form heterodimers and accelerates their degradation, thereby blocking ODC activity and reducing intracellular polyamine synthesis; (2) binds to the polyamine transport carrier on the cell membrane, thereby inhibiting the intracellular transfer of polyamines from the extracellular environment. The intracellular AZ activity is simultaneously regulated by another factor, anti-enzyme inhibitor (AZIN), which forms heterodimers with AZ with higher affinity, thereby competitively releasing the ODC captured by AZ and restoring its activity. Furthermore, the intracellular polyamine content is another important factor affecting the activity of AZ, and high polyamine concentrations enable the synthesis of functional AZ protein molecules by inducing a specific frameshift translation mechanism. Therefore, an ODC (optical Density-dependent protein) regulation network taking AZ-AZIN as a core exists in cells, and the ODC regulation network is an ideal polyamine regulation molecular target.
Studies at a cellular level have found that ODC inhibitors represented by Difluoromethylornithine (DFMO) can deplete intracellular polyamines by blocking ODC activity, thereby inhibiting tumor cell proliferation and inducing tumor cell apoptosis. However, these direct ODC-targeting inhibitors are not clinically effective when used as antitumor drugs because they require high doses and thus cause adverse effects that are difficult for patients to endure, and because when the synthesis of polyamines in cells is inhibited, polyamines in the extracellular environment can enter cells through polyamine transport vectors (polyamine transport) on the cell membranes to compensate for the decrease in the polyamine content in cells caused by the inhibition of synthesis. Therefore, a new method and a new way for inhibiting the ODC activity are explored, so that the method becomes a research hotspot in the field and has important clinical significance and application prospect.
Disclosure of Invention
The invention aims to design and screen an AZIN inhibitor by using AZ and AZIN in the regulation network as molecular targets and applying a computer-aided drug design technology, so as to interfere the interaction of AZ-AZIN, reduce the levels of ODC and polyamine in cells and be used for preparing drugs for treating tumors.
The specific structural formula of the small molecule inhibitor AZIN18 is as follows:
the small molecule inhibitor AZIN18 is applied to the preparation of drugs for inhibiting anti-enzyme inhibitors.
The small molecule inhibitor AZIN18 is applied to the preparation of drugs for inhibiting human prostate cancer.
The application of the preparation of the medicament for inhibiting the human prostate cancer, in particular to the application of the preparation of the medicament for inhibiting the growth and the propagation of the human prostate cancer PC3 cells.
Drawings
FIG. 1 screens pharmacophore models of AZIN small molecule inhibitors.
FIG. 2 immunoblotting was performed to detect changes in the amounts of ODC, AZ-1 and AZIN-1 proteins in PC3 cells.
Fig. 3 HPLC assay of the polyamine content in PC3 cells: p <0.01, x: p < 0.05.
FIG. 4 MTT method for detecting the growth inhibition effect of small molecule drug AZIN18 on PC3 cells at different times.
FIG. 5 flow cytometry was used to examine the effect of small molecule drug AZIN18 and control on the growth cycle of PC3 cells.
FIG. 6 flow cytometry is used to detect the effect of AZIN18 and control group, respectively, on the induction of apoptosis in PC3 cells.
Detailed Description
A small molecule inhibitor AZIN18 has a specific structural formula as follows:
results of pharmacophore screening
Pdb was the initial structure of AZIN crystal structure (3 btn), and a pocket detection module was used to search for possible binding sites in the receptor, finding a total of 5 possible binding sites. Binding AZ to AZIN. Mutation data of the interface, the pocket containing the most bound interface residues was selected for further screening of small molecule inhibitors. Based on the key amino acid residue characteristics of the determined active site, a pharmacophore model of the binding pocket was constructed using the pharmacophore method (FIG. 1). And searching and screening a SPECS compound database by using the constructed pharmacophore model, and finally purchasing the AZIN18 compound.
Effect of Small molecule inhibitor AZIN18 on the levels of ODC, AZ, AZIN and polyamines in PC3 cells
1. Immunoblotting method for detecting changes of ODC, AZ-1 and AZIN-1 protein contents in PC3 cells
Drug-treated cells and control cells were collected, lysed with RIPA cell lysate for half an hour, centrifuged at 12000rmp by a centrifuge to collect proteins, and then protein was quantified by BCA method. After subsequent electrophoretic separation of equal amounts of protein in each group, the proteins were electrically transferred to PVDF membrane, blocked with 5% skim milk (20mmol/L Tris-HCl,150mmol/L NaCl, 0.05% Tween-20, pH7.4) for one hour, incubated overnight with anti-ODC, AZIN-1 and AZ-1 monoclonal antibodies, and then incubated with polyclonal antibodies for one hour at room temperature. Protein expression was detected on ECL machines. As shown in FIG. 2, the ODC protein content was significantly reduced compared to the control cells, suggesting that AZIN18 was able to effectively interfere with the interaction between AZ and AZIN proteins, accelerating the degradation of ODC, and thus reducing the ODC protein content in the cells.
HPLC detection of Effect of AZIN18 on polyamine content in human prostate cancer PC3 cells
The drug-treated cells and control cells were collected, lysed with RIPA cell lysate to obtain proteins, followed by extraction of polyamines through benzoylation, and detection of polyamine content by Waters 2695 type high performance liquid chromatography, 2489UV/Vis type array detector and MYC column (150mmx4.6mm, 5 μm) with acetonitrile-water (40: 60), flow rate 1.0mL/min, 254nm and room temperature (column temperature). The results show that the levels of putrescine and spermine were reduced in cells treated with AZIN18 compared to control cells, indicating that AZIN18 interferes with cellular polyamine metabolism, as shown in figure 3.
Research on antitumor activity of small molecule inhibitor AZIN18
AZIN18 is effective in inhibiting growth and reproduction of human prostate cancer PC3 cells
Taking PC3 cells in logarithmic growth phase at 4X 103After culturing for 24h, 1640 medium containing the small molecule drug AZIN18 was added to each well of the 96-well cell culture plate at each well to give final concentrations of AZIN18 of 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M and 100. mu.M (4 duplicate wells per set). There are no-drug control group and blank group at the same time. After further culturing for 24h, 48h and 72h respectively, removing the cell culture medium in the culture plate, adding MTT reagent with the final concentration of 0.2g/L, incubating for 4h at 37 ℃, adding 150 mu L of DMSO, shaking and mixing uniformly, and measuring the absorbance value at 570 nm. The cell proliferation inhibition rate was (a control well-a experimental well)/a control well × 100%.
The MTT method detects the inhibition effect of SLD9059 on A549 cells, and the result shows that all concentrations of AZIN18 can obviously inhibit the growth of PC3 cells (P <0.001), and the inhibition effect on the growth of PC3 is increased along with the increase of the concentration of the medicament and the inhibition effect is increased along with the increase of time (P <0.001) (figure 4).
Antitumor action mechanism of small molecule inhibitor AZIN18
AZIN18 to alter the growth cycle of human prostate carcinoma PC3 cells
Taking PC3 cells in logarithmic growth phase at 1.3X 105The concentration of each well is inoculated on a 6-well cell culture plate, after 24 hours of culture, 1640 culture medium containing a small molecule drug AZIN18 is added into the cell wells, so that the final concentration of AZIN18 is 42.5 mu M, and a non-drug control group is arranged at the same time, and both groups are treated for 48 hours. Cells were processed according to the cell cycle detection kit instructions and cell cycle was detected by flow cytometry. The results show that: AZIN18 caused a G0/G1 phase arrest in PC3 cells (FIG. 5), increasing from 64.16% to 75.4% (P)<0.05)。
AZIN18 Induction of apoptosis in human prostate carcinoma PC3 cells
Taking PC3 cells in logarithmic growth phase at 1.3X 105The concentration of each well is inoculated on a 6-well cell culture plate, after 24 hours of culture, 1640 culture medium containing a small molecule drug AZIN18 is added into the cell wells, so that the final concentration of AZIN18 is 42.5 mu M, and a non-drug control group is arranged at the same time, and both groups are treated for 48 hours. According to the description of the apoptosis kitRegulating cells, and detecting the apoptosis condition by a flow cytometer. The results are shown in FIG. 6: compared with the control cells, the ratio of early apoptotic cells (annexin V +/PI-) is increased from 1.15% to 38.8%, and the ratio of late apoptotic cells (annexin V +/PI +) is increased from 1.87% to 19.3%, indicating that AZIN18 can effectively induce PC3 cells to undergo apoptosis.
Claims (3)
1. The application of a small molecule inhibitor AZIN18 in preparing a medicament for inhibiting an anti-enzyme inhibitory factor AZIN is characterized in that the structural formula of the small molecule inhibitor is as follows:
2. the use of the small molecule inhibitor AZIN18 according to claim 1 for the preparation of a medicament for the inhibition of human prostate cancer.
3. The use according to claim 2, wherein the small molecule inhibitor AZIN18 is used for the preparation of a medicament for inhibiting the growth and proliferation of human prostate cancer PC3 cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910273003.5A CN109942503B (en) | 2019-04-04 | 2019-04-04 | Small molecule inhibitor AZIN18 and application thereof in pharmacy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910273003.5A CN109942503B (en) | 2019-04-04 | 2019-04-04 | Small molecule inhibitor AZIN18 and application thereof in pharmacy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109942503A CN109942503A (en) | 2019-06-28 |
| CN109942503B true CN109942503B (en) | 2022-03-04 |
Family
ID=67013765
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910273003.5A Active CN109942503B (en) | 2019-04-04 | 2019-04-04 | Small molecule inhibitor AZIN18 and application thereof in pharmacy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109942503B (en) |
-
2019
- 2019-04-04 CN CN201910273003.5A patent/CN109942503B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| AZ/AZIN调控对A549非小细胞肺癌细胞作用研究;欧洁 等;《中国生物化学与分子生物学会第十二届全国会员代表大会暨2018年全国学术会议摘要集》;20181025;第76页 * |
| siＲNA靶向抗酶抑制剂下调前列腺癌细胞PC3中鸟氨酸脱羧酶表达;金涛 等;《基础医学与临床》;20180402;第38卷(第4期);第475-479页 * |
| STN Registry 数据库;审查员;《STN检索报告》;20040802;第1页 * |
| 鸟氨酸脱羧酶抗酶-1过表达促进非小细胞肺癌细胞系A549凋亡;欧洁 等;《基础医学与临床》;20190305;第39卷(第3期);第332-336 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109942503A (en) | 2019-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sun et al. | Functional metabolomics discover pentose and glucuronate interconversion pathways as promising targets for Yang Huang syndrome treatment with Yinchenhao Tang | |
| Zia et al. | Identification of potential TNF-α inhibitors: from in silico to in vitro studies | |
| Spurny et al. | Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines | |
| Kalid et al. | Consensus Induced Fit Docking (cIFD): methodology, validation, and application to the discovery of novel Crm1 inhibitors | |
| Qiu et al. | Dissect new mechanistic insights for geniposide efficacy on the hepatoprotection using multiomics approach | |
| Yang et al. | GalaxySagittarius: structure-and similarity-based prediction of protein targets for druglike compounds | |
| Ammarah et al. | Identification of new inhibitors against human Great wall kinase using in silico approaches | |
| Wang et al. | Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication | |
| CN109988086B (en) | Small molecule inhibitor AZIN30 and application thereof in pharmacy | |
| Margolles‐Clark et al. | Effective and specific inhibition of the CD40–CD154 costimulatory interaction by a naphthalenesulphonic acid derivative | |
| Zhu et al. | ADME/toxicity prediction and antitumor activity of novel nitrogenous heterocyclic compounds designed by computer targeting of alkylglycerone phosphate synthase | |
| Mi et al. | A network pharmacology study on analgesic mechanism of Yuanhu-Baizhi herb pair | |
| Heusser et al. | Functional validation of virtual screening for novel agents with general anesthetic action at ligand-gated ion channels | |
| Triveri et al. | Protein allostery and ligand design: Computational design meets experiments to discover novel chemical probes | |
| Gao et al. | Tetrahydroxy stilbene glycoside attenuates acetaminophen‐induced hepatotoxicity by UHPLC‐Q‐TOF/MS‐based metabolomics and multivariate data analysis | |
| CN109942503B (en) | Small molecule inhibitor AZIN18 and application thereof in pharmacy | |
| CN109988135B (en) | Small molecule inhibitor AZIN19 and application thereof in pharmacy | |
| CN109942467B (en) | Small molecule inhibitor AZIN32 and application thereof in pharmacy | |
| Fang et al. | Study on the potential mechanism of Semen Strychni against myasthenia gravis based on network pharmacology and molecular docking with experimental verification | |
| He et al. | In silico approaches using pharmacophore model combined with molecular docking for discovery of novel ULK1 inhibitors | |
| Zhang et al. | Virtual Screening Inhibitors of Ubiquitin‐specific Protease 7 Combining Pharmacophore Modeling and Molecular Docking | |
| Kumar et al. | Identification of novel inhibitors of human Chk1 using pharmacophore-based virtual screening and their evaluation as potential anti-cancer agents | |
| Jiang et al. | Discovery of potential negative allosteric modulators of mGluR5 from natural products using pharmacophore modeling, molecular docking, and molecular dynamics simulation studies | |
| Ma et al. | Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type‐B receptor 4 | |
| Yang et al. | Combined pharmacophore models as virtual screening protocol against BRD4 (1) inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |