CN109942467B

CN109942467B - Small molecule inhibitor AZIN32 and application thereof in pharmacy

Info

Publication number: CN109942467B
Application number: CN201910272540.8A
Authority: CN
Inventors: 孙丽丹; 谢晓兰; 翁文婷; 吕凤娇
Original assignee: Quanzhou Normal University
Current assignee: Quanzhou Normal University
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2020-09-25
Anticipated expiration: 2039-04-04
Also published as: CN109942467A

Abstract

The invention provides a small molecular inhibitor AZIN32, which has a structural formula as follows:

the small molecule inhibitor AZIN32 can be applied to preparation of drugs for inhibiting anti-enzyme inhibitory factors, and AZIN32 can inhibit proliferation of human prostate cancer PC3 cells, reduce the contents of ODC protein and polyamine in the cells, change the growth cycle of the PC3 cells, and induce the PC3 cells to undergo apoptosis.

Description

Small molecule inhibitor AZIN32 and application thereof in pharmacy

Technical Field

The invention relates to the field of biological medicines, in particular to a small molecular inhibitor AZIN32 and application thereof in pharmacy.

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 carriers (polyamineransports) on cell membranes and 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 an ODC regulation network as molecular targets and applying a computer-aided drug design technology, so as to interfere the interaction of AZ-AZIN, reduce the level of ODC and polyamine in cells and be used for preparing drugs for treating tumors.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an AZIN inhibitor is named as a small molecule inhibitor AZIN32, and has a specific structural formula as follows:

the small molecule inhibitor AZIN32 is applied to the preparation of drugs for inhibiting anti-enzyme inhibitors.

The small molecule inhibitor AZIN32 is applied to the preparation of drugs for inhibiting human prostate cancer.

Furthermore, the medicine is a medicine for inhibiting the growth and the reproduction of the human prostate cancer PC3 cells.

The invention provides a small molecular inhibitor AZIN32 for inhibiting an enzyme-resistant inhibitory factor, which can be used for preparing a medicament for treating tumor diseases, can effectively interfere the interaction between AZ and AZIN proteins, accelerates the degradation of ODC, reduces the content of ODC protein in cells, interferes the metabolism of polyamine in the cells, and can obviously inhibit the growth of PC3 cells, and the application of the small molecular inhibitor AZIN32 in pharmacy has important clinical significance and application prospect.

Drawings

FIG. 1 is a pharmacophore model for screening AZIN small molecule inhibitors.

FIG. 2 shows the immunoblotting of PC3 to detect the change of ODC, AZ-1, AZIN-1 protein content in cells.

Fig. 3 shows HPLC assay of PC3 intracellular polyamine content: p <0.01, x: p < 0.05.

FIG. 4 shows that MTT method detects the growth inhibition of PC3 cells by small molecule drug AZIN 32.

FIG. 5 shows the effect of AZIN32 as a small molecule drug on the growth cycle of PC3 cells detected by flow cytometry.

FIG. 6 shows that small molecule drug AZIN32 induces PC3 apoptosis by flow cytometry.

The specific implementation mode is as follows:

example 1

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). Searching and screening a SPECS compound database by using the constructed pharmacophore model, and finally screening to obtain a compound named as a small molecule inhibitor AZIN32, wherein the compound can be purchased and obtained, and the specific structural formula is as follows:

example 2

Effect of Small molecule inhibitor AZIN32 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

The cells treated with the drug AZIN32 and the control cells were collected, lysed with RIPA cell lysate for half an hour, centrifuged at 12000 rpm in a centrifuge to collect the protein, and the protein was quantified by BCA. 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 AZIN32 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 AZIN32 on polyamine content in human prostate cancer PC3 cells

Cells treated with the drug AZIN32 and cells of the control group were collected, after obtaining proteins by lysis with RAPA cell lysate, polyamines were extracted by benzoylation, and polyamine content was detected by Waters 2695 type high performance liquid chromatograph, 2489UV/Vis type array detector and MYC column (150mmx4.6mm, 5 μm) under acetonitrile-water (40: 60), flow rate 1.0mL/min, 254nm and room temperature (column temperature). The results show that the levels of putrescine, spermine and spermidine were reduced in cells treated with AZIN32 compared to control cells, indicating that AZIN32 interferes with cellular polyamine metabolism, as shown in figure 3.

Example 3

Research on antitumor activity of small molecule inhibitor AZIN32

AZIN32 is effective in inhibiting growth and reproduction of human prostate cancer PC3 cells

Taking PC3 cells in logarithmic growth phase at 4 × 10³And inoculating 96-well cell culture plates at each well, after culturing for 24h, respectively adding 1640 culture medium containing a small-molecule drug AZIN32 into the wells to enable the final concentrations of AZIN32 to be 0 mu M, 12.5 mu M, 25 mu M, 50 mu M, 75 mu M and 100 mu M (each group is provided with 4 multiple wells), simultaneously arranging a non-drug-added control group and a blank group, respectively after continuously culturing for 24h, 48h and 72h, removing the cell culture medium in the culture plates, adding an MTT reagent at the final concentration of 0.2g/L, incubating for 4h at 37 ℃, adding 150 mu L of DMSO, shaking and uniformly mixing, and measuring the absorbance value at 570nm, wherein the cell proliferation inhibition rate is (A control well-A experimental well)/A control well × 100%.

The results show that all concentrations of AZIN32 significantly inhibited the growth of PC3 cells (P <0.01), and that the inhibition of the growth of PC3 increased with increasing concentration of the drug and with increasing time (P <0.05) (fig. 4).

Example 4

Research on anti-tumor action mechanism of small molecule inhibitor AZIN32

AZIN32 to alter the growth cycle of human prostate carcinoma PC3 cells

Taking PC3 cells in logarithmic growth phase at 1.3 × 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 AZIN32 is added into the cell wells, so that the final concentration of AZIN32 is 51.5 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: AZIN32 caused an increase in S-phase arrest in PC3 cells (FIG. 5), from 20.5% to 34.4%, respectively (P)<0.05)。

AZIN32 Induction of apoptosis in human prostate carcinoma PC3 cells

Taking PC3 cells in logarithmic growth phase at 1.3 × 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 AZIN32 is added into the cell wells, so that the final concentration of AZIN32 is 51.5 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 apoptosis kit instructions,and detecting the apoptosis condition by a flow cytometer. The results are shown in FIG. 6: compared with control cells, the number of annexin V-FITC/PI double-stained cells increased from 1.87% to 12.9%, suggesting that our AZIN32 effectively induces apoptosis of PC3 cells.

Based on the teaching of the above examples, the small molecule inhibitor AZIN32 can be applied to the preparation of drugs for inhibiting anti-enzyme inhibitory factors, and drugs for inhibiting human prostate cancer, such as drugs for inhibiting the growth and reproduction of human prostate cancer PC3 cells.

Claims

1. The application of a small molecule inhibitor AZIN32 in preparing a medicament for inhibiting an enzyme inhibitor is disclosed, wherein the structural formula of the small molecule inhibitor is as follows:

2. the application of a small molecule inhibitor AZIN32 in preparing a medicament for inhibiting human prostate cancer is disclosed, wherein the structural formula of the small molecule inhibitor is as follows:

3. the use of claim 2, wherein said medicament is a medicament for inhibiting the growth and proliferation of human prostate cancer PC3 cells.