CN109988086B - Small molecule inhibitor AZIN30 and application thereof in pharmacy - Google Patents
Small molecule inhibitor AZIN30 and application thereof in pharmacy Download PDFInfo
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- CN109988086B CN109988086B CN201910272991.1A CN201910272991A CN109988086B CN 109988086 B CN109988086 B CN 109988086B CN 201910272991 A CN201910272991 A CN 201910272991A CN 109988086 B CN109988086 B CN 109988086B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
- C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
Abstract
The invention provides a small molecular inhibitor AZIN30, which has a structural formula as follows:
the invention relates to application of a small molecular inhibitor AZIN30 in preparing a medicament for inhibiting an enzyme-resistant inhibitory factor. The results show that: AZIN30 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.
The invention content is as follows:
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 AZIN30 is as follows:
the small molecule inhibitor AZIN30 is applied to the preparation of drugs for inhibiting anti-enzyme inhibitors.
The small molecule inhibitor AZIN30 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 AZIN30 on PC3 cells.
FIG. 5 flow cytometry method for detecting the effect of small molecule drug AZIN30 on the growth cycle of PC3 cells.
FIG. 6 flow cytometry detection of the induction of PC3 apoptosis by the small molecule drug AZIN 30.
The specific implementation mode is as follows:
a small molecule inhibitor AZIN30 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. Combining mutation data for AZ and AZIN binding interfaces, the pocket containing the most binding 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 AZIN30 compound.
Effect of Small molecule inhibitor AZIN30 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. The results are shown in fig. 2, and compared with the control cells, the content of ODC protein is significantly reduced, suggesting that our AZIN30 can effectively interfere the interaction between AZ and AZIN protein, accelerate the degradation of ODC, and thus reduce the content of ODC protein in the cells.
HPLC detection of Effect of AZIN30 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 spermine content in cells treated with AZIN30 was reduced compared to control cells, indicating that AZIN30 interferes with cellular polyamine metabolism, as shown in FIG. 3.
Research on antitumor activity of small molecule inhibitor AZIN30
AZIN30 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 AZIN30 was added to each well of the 96-well cell culture plate at each well to give final concentrations of AZIN30 of 0. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M and 100. mu.M (4 duplicate wells per group). 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 AZIN30 can obviously inhibit the growth of PC3 cells (P <0.05), 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 <0.05) (figure 4).
Research on anti-tumor action mechanism of small molecule inhibitor AZIN30
AZIN30 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 AZIN30 is added into the cell wells, so that the final concentration of AZIN30 is 72 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: AZIN30 caused S-phase arrest in PC3 cells (FIG. 5), increasing from 21.5% to 35.78% (P)<0.05)。
AZIN30 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 AZIN30 is added into the cell wells, so that the final concentration of AZIN30 is 72 mu M, and a non-drug control group is arranged at the same time, and both groups are treated for 48 hours. Treating the cells according to the instructions of the apoptosis kit, anddetecting the apoptosis condition by a flow cytometer. The results are shown in FIG. 6: compared with the control cells, the ratio of early apoptosis cells (annexin V +/PI-) is increased from 1.15% to 38.6%, and the ratio of late apoptosis cells (annexin V +/PI +) is increased from 1.87% to 12.3%. It was suggested that AZIN30 was effective in inducing apoptosis in PC3 cells.
Claims (2)
1. The application of a small molecule inhibitor AZIN30 in preparing a medicament for inhibiting an enzyme inhibitor is characterized in that the structural formula of the small molecule inhibitor is as follows:
2. the application of a small molecule inhibitor AZIN30 in preparing a medicament for inhibiting the growth and the reproduction of human prostate cancer PC3 cells is characterized in that the structural formula of the small molecule inhibitor is as follows:
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| CN101948541A (en) * | 2005-06-20 | 2011-01-19 | 健泰科生物技术公司 | Compositions and methods for the diagnosis and treatment of tumor |
| CN102964279A (en) * | 2012-12-12 | 2013-03-13 | 中国药科大学 | Kappa-opioid receptor agonists with peripheral analgesic effect |
| CN103491956A (en) * | 2010-09-13 | 2014-01-01 | 麦克罗拜奥提斯公司 | Inhibitors of viral entry into mammalian cells |
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| US9790233B2 (en) * | 2012-04-16 | 2017-10-17 | Case Western Reserve University | Compositions and methods of modulating 15-PGDH activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948541A (en) * | 2005-06-20 | 2011-01-19 | 健泰科生物技术公司 | Compositions and methods for the diagnosis and treatment of tumor |
| CN103491956A (en) * | 2010-09-13 | 2014-01-01 | 麦克罗拜奥提斯公司 | Inhibitors of viral entry into mammalian cells |
| CN102964279A (en) * | 2012-12-12 | 2013-03-13 | 中国药科大学 | Kappa-opioid receptor agonists with peripheral analgesic effect |
Non-Patent Citations (4)
| Title |
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| 432019-57-3;CAS;《STN Registry 数据库》;20020619;1-4 * |
| Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic−Industry Partnership;Charlie Y Mo et al;《ACS Infect. Dis》;20171224;第4卷;349-359 * |
| 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》;20180430;第38卷(第4期);475-479 * |
| 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》;20190331;第39卷(第3期);332-336 * |
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