CN112957357B - Target KLF4 ubiquitination small molecule inhibitor and application thereof - Google Patents
Target KLF4 ubiquitination small molecule inhibitor and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine and molecular therapy, and particularly relates to a target KLF4 protein ubiquitination small-molecule inhibitor and application thereof. A small molecule inhibitor targeting KLF4 ubiquitination has a structural general formula as follows:
the small molecule inhibitor can inhibit the ubiquitination of KLF4 and increase the expression of KLF4 protein, thereby inhibiting the proliferation of gastric cancer cells. The KLF4 ubiquitination-targeted small molecule inhibitor, the pharmaceutically acceptable salt, the hydrate or the solvate thereof and the pharmaceutical composition are applied to preparation of drugs for treating gastric cancer. The small-molecule inhibitor and the pharmaceutical composition of the small-target KLF4 ubiquitination can be used for inhibiting KLF4 ubiquitination, have high selection specificity and strong pertinence, have small IC50, and prove that the affinity is strong, the clinical use effect is good, and the remission rate is high. The KLF4 ubiquitination-targeted small-molecule inhibitor plays an important role in the field of gastric cancer molecular targeted therapy, and provides a new targeted therapeutic drug for clinical therapy of gastric cancer.
Description
Technical Field
The invention belongs to the fields of biological medicines and molecular therapy, and particularly relates to a small-molecule inhibitor targeting the ubiquitination of KLF4 protein (Krluppel-like factor4) and application thereof in inhibiting and killing gastric cancer cells and the like.
Background
Cancer is one of the diseases seriously threatening human health in the 21 st century. Malignant tumors are the most mortality disease. Statistically, one out of every four people in the world is at risk for cancer. Currently, the number of cancer patients in the world increases year by year and there is a trend toward younger patients. The global trend towards increased cancer morbidity and mortality suggests that cancer will become the first killer in humans and have posed a significant public health challenge. Improving the prognosis of cancer patients is a key factor in increasing patient survival. Tumor generation is a process in which multiple mechanisms are involved together, and multiple proteins are abnormally expressed. Therefore, the research and development of drugs aiming at the cancer target is an urgent problem to be solved.
The ubiquitin-protease system is one of the pathways for protein degradation, selectively degrading the intended target protein. The ubiquitin-proteasome not only degrades proteins, but also can participate in regulation and control of biological processes such as cell proliferation and cell cycle. In addition, the ubiquitin pathway regulates a variety of cellular processes such as autophagy, apoptosis, signal transduction, receptor endocytosis control, antigen processing, degradation of antiviral proteins, and viral proliferation. The intracellular ubiquitin-proteasome pathway is composed of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E1, ubiquitin ligase E3 and 26S protease compound, the protein ubiquitination degradation is completed in two steps, namely, the cascade reaction ubiquitination modification substrate protein composed of E1-E2-E3 is firstly degraded by proteasome. It has now been found that ubiquitination regulates protein degradation and plays an important role in the development of tumors.
KLF4 can act on different target genes in different tissues to exert cancer-inhibiting or cancer-promoting effects, and mainly play a cancer-inhibiting role in gastrointestinal tumors. The intracellular action of KLF4 is tightly regulated by the ubiquitin-proteasome pathway, Ubiquitination (Ubiquitination) being an important pathway for post-translational modification of KLF 4. The key link of KLF4 in playing its transcriptional regulation role is the inhibition of KLF4 ubiquitination and promotion of nuclear translocation. Research has shown that the F-box family (FBXW, FBXL and FBXO) can mediate the recognition and recruitment of ubiquitin substrates through the formation of SCF (Skp1-Cullin1-F-box) complex of E3 ligase, thereby playing an important role in protein ubiquitination and cancer development. The F-box family proteins such as FBXO22, FBXO32, SKP2 and the like are predicted to be targeted and combined with an E3 potential recognition site, zf-H2C2_2 domain in a KLF4 protein sequence, so that KLF4 ubiquitination is promoted, the expression level of the KLF4 in a tumor is inhibited, and the occurrence and development of the tumor are promoted.
At present, no small-molecule inhibitor aiming at the ubiquitination of the KLF4 is disclosed in the prior art, so that the development and research of a targeted therapeutic medicament aiming at the KLF4 protein aiming at the KLF4 and the ubiquitination thereof become a problem to be solved urgently at present.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a KLF4 ubiquitination inhibitor and application thereof in inhibiting and killing gastric cancer cells, and the invention aims to design a KLF4 ubiquitination inhibitor aiming at a KLF4 ubiquitination site, the inhibitor has high affinity to KLF4, has strong specificity to KLF4 at a nanomolar level, has a good inhibition effect on ubiquitination of KLF4, so that the expression of KLF4 protein is increased, and the inhibitor has a certain effect on treatment of gastric cancer. According to the invention, a micromolecule targeted drug aiming at a zf-H2C2_2 structural domain of KLF4 protein is screened out through high-throughput drug screening, and the compound is structurally modified according to the Ribes-King principle, so that the solubility of the drug is better, the affinity to KLF4 is stronger, and finally, a brand-new micromolecule inhibitor LK-SI3A aiming at KLF4 ubiquitination is obtained. The IC50 value of LK-SI3A on gastric cancer cells is detected through a CCK8 experiment, the inhibition effect of LK-SI3A on gastric cancer cells is detected through a plate clone forming experiment, the effective compound is determined to aim at a KLF4 target through Western blot, and further the value and significance of continuously researching a small molecule inhibitor aiming at KLF4 ubiquitination are achieved.
In order to achieve the above object, the present invention adopts the following technical solutions.
A small molecule inhibitor targeting KLF4 ubiquitination has a structural general formula as follows:
further, the structural formula of the KLF4 ubiquitination-targeted small molecule inhibitor LK-SI3A is as follows:
furthermore, the small molecule inhibitor can inhibit the ubiquitination of KLF4 and increase the expression of KLF4 protein, thereby inhibiting the proliferation of gastric cancer cells.
A pharmaceutical composition, which comprises the above-mentioned KLF4 ubiquitination-targeted small molecule inhibitor, its pharmaceutically acceptable salt, hydrate or solvate, and a pharmaceutically acceptable carrier.
The KLF4 ubiquitination-targeted small molecule inhibitor, the pharmaceutically acceptable salt, the hydrate or the solvate thereof and the application of the pharmaceutical composition in preparing a medicine for treating gastric cancer are provided.
The medicament is in any pharmaceutically acceptable dosage form.
Preferably, the pharmaceutical dosage form is an injectable formulation.
The medicament is in any pharmaceutically acceptable dose.
Compared with the prior art, the invention has the following beneficial effects.
According to the invention, a micromolecule targeted drug aiming at a zf-H2C2_2 structural domain of KLF4 protein is screened out through high-throughput drug screening, and the compound is structurally modified according to the Ribes-King principle, so that the solubility of the drug is better, the affinity to KLF4 is stronger, and finally, a brand-new micromolecule inhibitor LK-SI3A aiming at KLF4 ubiquitination is obtained. The IC50 value of LK-SI3A on gastric cancer cells is detected through a CCK8 experiment, the inhibition effect of LK-SI3A on gastric cancer cells is detected through a plate clone forming experiment, the effective compound is determined to aim at a KLF4 target through Western blot, and further the value and significance of continuously researching a small molecule inhibitor aiming at KLF4 ubiquitination are achieved.
The small-molecule inhibitor and the pharmaceutical composition of the small-target KLF4 ubiquitination can be used for inhibiting KLF4 ubiquitination, have high selection specificity and strong pertinence, have small IC50, and prove that the affinity is strong, the clinical use effect is good, and the remission rate is high. The KLF4 ubiquitination-targeted small-molecule inhibitor plays an important role in the field of gastric cancer molecular targeted therapy, and provides a new targeted therapeutic drug for clinical therapy of gastric cancer.
Drawings
FIG. 1 is a schematic diagram of the binding 3D model and score of KLF4 ubiquitination inhibitor LK-SI3A and Arg473 of human KLF4 protein ubiquitination domain zf-H2C 2.
FIG. 2 is a schematic diagram of the 2D model of the binding of KLF4 ubiquitination inhibitor LK-SI3A to Arg473 of the ubiquitination domain zf-H2C2 of human KLF4 protein.
FIG. 3 shows the killing effect of LK-SI3A on gastric cancer cell HGC-27 and IC50 value.
FIG. 4 shows the effect of LK-SI3A on the proliferation inhibition of gastric cancer cell HGC-27.
FIG. 5 is the regulatory effect of LK-SI3A on the ubiquitination of KLF4 protein.
FIG. 6 shows that LK-SI3A increased KLF4 protein level to induce apoptosis of gastric cancer cell HGC-27.
Detailed Description
The following detailed description of embodiments of the invention is provided in connection with the accompanying drawings and examples. The following examples illustrate the invention in detail, but are not intended to limit the scope of the invention. This example was performed using routine experimentation, as is well known to those skilled in the art, and can be performed according to the present example using the instructions provided by the manufacturer of the materials.
Examples are given.
1. Test materials.
1.1 cell line.
Human gastric carcinoma cell line HGC-27.
1.2 test reagents.
RPMI-1640 medium, trypsin, fetal bovine serum, 0.5% crystal violet, and CCK8 reagent.
2. Experimental methods.
2.1 MOE software for drug screening.
Firstly, the protein structure of KLF4 is modeled by MOE homology, molecular docking is carried out after structure optimization, and finally LK-SI3A (synthesized by Yanming) with better score is screened out, wherein the structural formula is as follows:
2.2 cell proliferation CCK8 experiments.
The Cell Counting Kit-8 (CCK-8 for short) reagent can be used for simple and accurate Cell proliferation and toxicity analysis. The basic principle is as follows: this reagent contained WST-8(2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt)). WST-8 can be reduced by a dehydrogenase in a cell to a yellow formazan product with high water solubility. Amount of formazan product formedProportional to the number of living cells. In this experiment, the stomach cancer cell HGC-27 in logarithmic growth phase was inoculated with 4X 10 cells3The density of individual cells/well was seeded onto 96-well plates. Then, the cells were treated with different concentrations of LK-SI3A for 24 hours, followed by 100. mu.l of fresh culture medium, and 10. mu.l of CCK8(Dojindo Molecular Technologies Inc., Japan) was added to each well for 1 hour. The absorbance at 450nm was measured using a multimode reader microplate reader (LD942, Beijing, China). Each group was set with 6 duplicate wells, and the control group was added with the same amount of medium while blank wells without cells and test substance were set. Cell viability ═ 100% x (experimental well OD value-blank well OD value)/(control group OD value-blank well OD value).
2.4 cell plate clone formation experiments.
The HGC-27 cell line in the logarithmic growth phase was carefully washed three times with 1 XPBS buffer, the cells were digested with an appropriate amount of trypsin, counted on a counting plate, and the cells were diluted to 500 cells/group with the medium. 500 cells were added to a medium containing 2mL, mixed well, added to a six-well plate, and LK-SI3A was added to a final concentration of 0.4. mu.M, and the cells were placed in a cell incubator to be cultured while maintaining equilibrium. After 7 days of continuous culture, the cells were observed for colony formation and replaced, i.e., after three careful washes with 1 XPBS buffer, the culture was continued by adding fresh medium, and after 14 days, the plates were removed. The medium was removed, washed carefully 2 times with 1 × PBS buffer and fixed with 4% paraformaldehyde for 20 min. Carefully wash twice with 1 × PBS buffer, add hematoxylin to cover the cells, and stain for 30 min. After final two careful washes with 1 × PBS buffer, cell colony formation can be observed and imaged for later statistical observation.
2.5 cell transfection and Western Blot.
And (4) selecting proper density according to the growth speed of the cells to pave the dish. The cell confluence before transfection reaches 70-80%, and the confluence degree after 2 days of transfection is about 90%. The culture medium does not contain antibiotics. The KLF4 plasmid was centrifuged at 3000g, and the mixed bacteria were centrifuged to the lower layer, and the supernatant was used (or the bacteria were precipitated with 75% ethanol). Preparation of transfection Using a 1mL system as an example: mu.L serum-free medium + lipo 2. mu.L, gently mixed, and left at room temperature for 5 min. 500 μ L serum-free medium + plasmid 2000ng, gently mixed, and left at room temperature for 5 min. The two tubes are combined, mixed evenly and placed for 20min, and the preparation of the transfection reagent is finished. The original medium was aspirated off, washed twice with 1 × PBS, the prepared transfection reagent was added, and the cell culture plate was gently shaken back and forth. Adding 400nM LK-SI3A into the cultured cells, culturing for 24h, extracting protein, quantifying the protein, performing protein denaturation, mixing with glue, loading, performing electrophoresis, transferring a membrane, sealing, incubating with a primary antibody and a secondary antibody, and finally performing chemiluminescence.
2.6 cell morphology observation.
LK-SI3A was added to the gastric cancer cell HGC-27 at a final concentration of 0.4. mu.M, and after 24 hours of culture, morphological photographs were taken using a microscope, as shown in FIGS. 1 to 6. FIG. 1 shows the 3D binding situation and site of KLF4 protein and LK-SI3A and KLF4 protein, which are KLF4 ubiquitination small molecule inhibitors, the binding energy of the LK-SI3A and KLF4 protein is-6.6051J/mol. FIG. 2 shows 2D binding of LK-SI3A to KLF4 protein. FIG. 3 shows that LK-SI3A shows the inhibition effect on gastric cancer cell HGC-27, with the increase of LK-SI3A concentration, the cell inhibition rate is increased, and the IC50 of LK-SI3A is 399.1 nM. FIG. 4 shows the inhibition of gastric cancer cell HGC-27 by LK-SI3A at a final concentration of 0.4. mu.M. FIG. 5 shows that addition of LK-SI3A at a final concentration of 0.4. mu.M promotes apoptosis of gastric cancer cell HGC-27. FIG. 6 shows primarily LK-SI3A causing scorch of gastric cancer cell HGC-27 by inhibiting the ubiquitination of KLF4 protein.
Claims (7)
1. A small molecule inhibitor targeting KLF4 ubiquitination is characterized in that the small molecule inhibitor is LK-SI3A, and the structural formula is as follows:
2. the small molecule inhibitor targeting KLF4 ubiquitination according to claim 1, wherein the small molecule inhibitor is capable of inhibiting KLF4 ubiquitination, increasing KLF4 protein expression, and further inhibiting gastric cancer cell proliferation.
3. A pharmaceutical composition comprising the KLF4 ubiquitinated targeted small molecule inhibitor of claim 1, or a pharmaceutically acceptable salt, hydrate thereof, and a pharmaceutically acceptable carrier.
4. Use of the KLF4 ubiquitination-targeted small molecule inhibitor of claim 1 or a pharmaceutically acceptable salt, hydrate thereof and the pharmaceutical composition of claim 3 for the preparation of a medicament for the treatment of gastric cancer.
5. The use of claim 4, wherein the medicament is in any pharmaceutically-therapeutically acceptable dosage form.
6. The use of claim 5, wherein the medicament is in the form of an injectable formulation.
7. The use of claim 4, wherein the medicament is in any pharmaceutically acceptable dose.
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| CN101248072A (en) * | 2005-05-25 | 2008-08-20 | 吉倪塞思技术公司 | 2-indolyl imidazo[4,5-d]phenanthroline derivatives and their use in the treatment of cancer |
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| JP5959537B2 (en) * | 2011-01-28 | 2016-08-02 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Substituted pyridinyl-pyrimidines and their use as pharmaceuticals |
| CN106543185B (en) * | 2016-11-10 | 2017-12-15 | 吉林大学 | A kind of compound for targetting ubiquitination degraded PLK1 and BRD4 albumen and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101248072A (en) * | 2005-05-25 | 2008-08-20 | 吉倪塞思技术公司 | 2-indolyl imidazo[4,5-d]phenanthroline derivatives and their use in the treatment of cancer |
Non-Patent Citations (2)
| Title |
|---|
| Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway;Zhang Jie等;《American Journal of Translational Research》;20191231;第11卷(第7期);全文 * |
| Prognostic significance of KLF4 expression in gastric cancer;Hashimoto Isaya等;《Oncology Letters》;20171231;第13卷(第2期);全文 * |
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