CN115969855B

CN115969855B - Application of APY29 in preparation of RSK4 kinase inhibitor

Info

Publication number: CN115969855B
Application number: CN202211618395.2A
Authority: CN
Inventors: 马静; 柴佳; 杨艳茹; 范林妮; 王凯靖; 刘一雄; 彭阳
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2024-01-26
Anticipated expiration: 2042-12-15
Also published as: CN115969855A

Abstract

The invention belongs to the field of tumor drug research, and particularly relates to application of APY29 in preparation of an RSK4 kinase inhibitor. The test result shows that the APY29 can effectively inhibit the phosphorylation of the RSK4 and inhibit the activation of the RSK 4. In vivo test results prove that the APY29 can inhibit proliferation, growth and invasion and migration of the kidney cancer cells. APY29, used in conjunction with sunitinib, also significantly reduced renal clear cell carcinoma angiogenesis.

Description

Application of APY29 in preparation of RSK4 kinase inhibitor

Technical Field

The invention belongs to the field of tumor drug research, and particularly relates to application of APY29 in preparation of an RSK4 kinase inhibitor.

Background

Renal cell carcinoma (renal cell carcinoma, RCC) is a common malignancy of the urinary system, originates from tubular epithelial cells, accounts for about 80-90% of renal malignancies, is diverse in tissue type, and is mainly renal clear cell carcinoma (clear cell renal cell carcinoma, ccRCC), and is histologically characterized by blood vessel-rich. Angiogenesis in renal cell carcinoma is closely related to the development, progression and treatment of tumors. Current treatment of RCC is still based on surgical excision, and for metastatic or unresectable RCC, first-line treatment is to use the tyrosine kinase inhibitor sunitinib, etc. against angiogenesis-related VEGF/VEGFR (vascular endothelial growth factor/vascular endothelial growth factor receptor). However, the adverse reactions caused by the targeted drugs are more, about 70% of patients can have adverse reactions of hypertension or hypertension progression, and drug resistance can occur in about one year. Therefore, the research of related strategies for preventing and treating RCC angiogenesis has important clinical significance, and can provide new theoretical basis and effective supplement for targeted treatment.

RSK4 is one of the Ribosomal S6 Kinase (RSK) family members, encoded by 746 amino acids, and serves as a downstream molecule of the MAPK pathway, playing an important role in the proliferation, survival, and cycle progression of cells, and also plays an important role in the development and invasion and metastasis of some tumors such as esophageal cancer, endometrial cancer, and breast cancer. Therefore, it is important to investigate inhibitors specific for RSK4 kinase and to provide a new therapeutic approach for patients with advanced renal cell carcinoma.

Disclosure of Invention

In view of the above technical problems, the present invention provides the following technical solutions:

in a first aspect of the invention there is provided the use of APY29 in the preparation of an RSK4 kinase inhibitor.

Preferably, said APY29 is used for the preparation of an inhibitor of the phosphorylation of the ribosomal protein S6 of the substrate downstream of RSK 4.

In a second aspect, the invention provides the use of APY29 in the manufacture of a medicament for the treatment/prophylaxis of diseases associated with RSK4 kinase.

Preferably, the disease associated with RSK4 kinase is renal cancer.

Preferably, the kidney cancer is renal clear cell carcinoma.

Preferably, the APY29 is used for preparing a proliferation inhibitor of renal clear cell carcinoma.

Preferably, the APY29 is used to prepare a blocker of renal clear cell carcinoma invasion and migration.

In a third aspect of the invention, there is provided a medicament for the treatment/prophylaxis of diseases associated with RSK4 kinase, comprising said APY29.

Preferably, the drug comprises sunitinib.

In a fourth aspect, the invention provides the use of the medicament in the manufacture of a medicament for inhibiting abnormally active angiogenesis caused by renal clear cell carcinoma.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides the use of APY29 in the preparation of an RSK4 kinase inhibitor. The test result shows that the APY29 can effectively inhibit the phosphorylation of RSK4 and inhibit the activation of RSK4, and the inhibition rate of the APY29 to RSK4 kinase is 98.4%. In vivo test results prove that the APY29 can inhibit proliferation, growth and invasion and migration of the kidney cancer cells. APY29, used in conjunction with sunitinib, also significantly reduced abnormally active angiogenesis caused by renal clear cell carcinoma.

Drawings

FIG. 1 is the inhibition of RSK4 enzyme by APY29 at various concentrations;

FIG. 2 is a graph showing the effect of Westernblot detection of different concentrations of APY29 on the phosphorylation levels of substrates downstream of RSK 4;

FIG. 3 is the effect of each group on cell proliferation of the cell ACHN;

fig. 4 is ic50= 0.6233 μm of APY29 in ACHN cells;

FIG. 5 is a Transwell assay for the effect of APY29 on ACHN cell invasion; A. transwell experiments of APY29 on ACHN cell invasion; B. statistical data of Transwell experiments;

FIG. 6 is the effect of APY29 on ACHN cells after RSK4 knockdown; A. ACHN invasion migration ability is weakened after RSK4 is knocked down; B. statistical data of the attack migration experiment;

FIG. 7 is the inhibition of tumor growth in mice by each group;

FIG. 8 is the effect of sunitinib alone and APY29 in combination on HUVEC cell tube formation;

figure 9 is statistical data of the effect of sunitinib alone and APY29 in combination on HUVEC cell tube number.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention and are not limiting of the invention claims. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the description of the presently disclosed subject matter.

As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "comprising" is a technical term used in claim language to mean that the recited element is present, but other elements may be added and still form a construct or method within the scope of the recited claims.

The CAS number of the APY29 used in the invention is 1216665-49-4, and the structure is shown as formula I:

example 1

Inhibition of RSK4 enzyme by APY29

1. Experimental method

The test uses ADP-Glo method to detect the effect of APY29 on RSK4 enzyme.

The initial concentration of the compound to be tested is 10 mu M, and the compound is diluted by 5 times of gradient, so that 6 compound APY29 solutions with different concentrations are obtained.

The specific operation process is as follows:

(1) The RSK4 enzyme, RSK Substrate, kinase buffer III (5 x buffer), dithiothreitol (DTT, 0.1M) and ATP (10 mM) were thawed on ice and the above reagents were required to be placed on ice all the time throughout the experiment.

(2) Preparing 5 Xkinase buffer III into 1 Xbuffer with deionized water, and adding DTT with concentration of 50 μm in 1 Xbuffer;

(3) 1 μl/well of 5 Xtest compound APY29 was added to a white microplate, and the microplate was centrifuged at 1000r/min for 1 minute on a centrifuge;

positive control wells (pos.ctrl): 1 μl/well of compound dilution solvent;

blank control wells (Blank): 1 μl/well 1 Xbuffer.

(4) After complete thawing of the RSK4 enzyme, 1 Xkinase buffer III was used to dilute the RSK4 enzyme to 1 ng/. Mu.l and 2. Mu.l/well was added to the white microplate at 2ng of RSK4 enzyme per well; blank wells were added with 2 μl/well 1 Xbuffer; the step is carried out on ice, and after the addition is finished, the micro-pore plate is centrifuged for 1 minute at 1000r/min on a centrifuge;

(5) Preparing an RSK Substrate/ATP mixed solution:

RSK Substrate/ATP mix: 130. Mu.l of RSK Substrate (1 mg/ml) was taken and 3.25. Mu.l of 5mM ATP and 127. Mu.l of 2 Xbuffer (note here diluted in proportion) were added, at which time the ATP concentration in the mixture was 62.5. Mu.M and the RSK Substrate concentration was 0.5mg/ml; this step is performed on ice;

(6) Taking 2 mu l/hole RSK Substrate/ATP mixed solution into a white micro-pore plate, wherein the concentration of RSK Substrate is 0.2mg/ml, the concentration of ATP is 25 mu M, and centrifuging the micro-pore plate for 1 minute at 1000r/min after adding;

(7) After centrifugation, attaching a membrane to the microporous plate, compacting the membrane, and incubating for 1 hour at 25 ℃;

(8) ADP-GloTM reagent and kinase detection related reagent needed in the Promega kit are equilibrated to room temperature, and kinase detection buffer and kinase detection substrate are mixed for use according to the instructions.

(9) After the incubation is finished, 5 μl/well of ADP-GloTM reagent is added to the white microplate, and the microplate is centrifuged at 1000 rpm for 1 min and incubated at 25deg.C for 40 min;

(10) After the incubation is finished, 10 μl/well of Kinase Detection mixture is added to the microplate, and the microplate is centrifuged at 1000r/min for 1 min and incubated at 25deg.C for 30 min;

(11) After the incubation is finished, performing chemiluminescence detection on a plate reader, and reading a luminescence value (RLU);

(12) Enzyme inhibition rate calculation:

enzyme inhibition ratio (%) =100- (sample luminescence value-blank luminescence value)/(positive luminescence value-blank luminescence value) ×100%.

2. Experimental results

As shown in FIG. 1, the inhibition of RSK4 enzyme by APY29 was also enhanced with increasing concentration of APY29. Curve fitting was performed using GraphPad software to give an IC50 value of 55.1nM. The compound APY29 is shown to be capable of effectively inhibiting the phosphorylation of RSK4 and inhibiting the activation of RSK4, and is an effective RSK4 inhibitor.

Example 2

Effect of APY29 on the phosphorylation level of the substrate downstream of RSK4

1. Experimental method

The experiment uses APY29 with different concentrations to stimulate a renal cancer cell line, and Westernblot detects the effect of the phosphorylation level of a substrate downstream of RSK 4. The specific operation process is as follows:

(1) Protein extraction, using the well-known century RIPA lysate. The cell culture flask is placed on ice for 20min for lysis, and is shaken back and forth to be blown by a gun head. Cells were scraped with a cell scraper, transferred to an EP tube and centrifuged at 4 ℃. Protein quantification can be performed by pipetting 10-20. Mu.l. Adding loading buffer (prepared from 100. Mu.l RIPA lysate (strong), 1. Mu.l phosphatase inhibitor and 1. Mu.l protein inhibitor, all from Kangshiji reagent Co.), boiling for 5min, centrifuging at 12000rpm for 5min, and storing in ultra-low temperature refrigerator. Tissue protein extraction: grinding with liquid nitrogen, melting, transferring into EP tube, cracking on ice for 20min, and centrifuging at 4deg.C.

(2) Protein quantification, using the kang century BCA quantification kit. Two complex wells were made for each sample, and the average was taken as a standard curve and the sample concentration was calculated.

(3) Protein electrophoresis kits were prepared using a well-known century SDS-PAGE gel. The concentration of the separation gel is 8% and the concentration of the concentrated gel is 5%. The separation glue is 1.5cm away from the upper edge of the front glass plate, and the water injection glue is used for sealing. The water-sealed liquid is sucked up by filter paper, the concentrated glue is poured in, and a comb is inserted. The electrophoresis was stopped when bromophenol blue was run out at a constant voltage of 100V for 1 hour for 30 min.

(4) Transferring, namely soaking the glue, the filter paper and the foam cushion in a transfer buffer solution precooled at 4 ℃ for balancing for 10min, soaking the PVDF membrane in methanol for 5min, and transferring to the transfer buffer solution for soaking for 10min after full wetting. Mounting a film transfer clamp sequence: black plate, sponge, 3 layers of filter paper, glue, film, 3 layers of filter paper, sponge, white plate. Bubble removal is needed in each step, and the membrane is placed at one time. The film was constantly circulated in an ice bath for 100min at 300 mA.

(5) The mixture was blocked for 1h using a 5% nonfat dry milk room temperature shaker. Rinse 10min x 3 times with TBST.

(6) And (3) incubating for the first time, wherein the membrane and the adhesive joint face are always upwards when the antibody is incubated, and incubating overnight at the temperature of 4 ℃ through a shaking table. The next day was rinsed with TBST.

(7) The secondary antibody is incubated for 1h at room temperature. Rinse with TBST.

(8) Luminescence was used, the detection results were scanned with a chemiluminescent imager using the kanji chemiluminescent detection kit.

(9) Semi-quantitative gray scale analysis is carried out on the image by using Photoshop software, and the relative expression quantity of the protein is calculated.

2. Experimental results

As shown in FIG. 2, the concentrations of 1uM and 10uMAPY29 were used to stimulate the renal cancer cell lines, respectively, and different concentrations of APY29 were found to inhibit the phosphorylation of the downstream substrate of RSK4 by Westernblot detection, and down-regulate the expression of P-RPS6 (S235/S236).

Example 3

Effect of APY29 on renal cancer cells

1. Experimental method

In the experiment, 1uM and 10uMAPY29 are adopted to respectively stimulate a renal cancer cell line (ACHN), CCK8 is used for detecting the influence of APY29 on the proliferation of the renal cancer cells, transwell is used for detecting the influence of APY29 on the migration of the renal cancer cells, and meanwhile BI-D1870 is used as a positive control, and no drug is added as a Blank control (Blank).

1.1, CCK8 detection steps:

was performed using CCK-8 cell proliferation kit from Shanghai Tao Shu Biotechnology Co. The specific operation process is as follows:

(1) The 96-well plate was seeded with 100 μl of cell suspension per well, 2,000 cells per well.

(2) Culturing or administration of the drugs is performed for a suitable time according to the experimental requirements.

(3) 10. Mu.L of CCK-8 solution was added to each well and incubated at 37 ℃.

(4) The enzyme label instrument selects the wavelength of 450nm to measure the absorbance value. Growth curves were plotted with time and absorbance values.

1.2, transwell migration experiment steps:

(1) The cells are digested. Washed once with PBS, resuspended in serum-free medium and the cell density was adjusted to 5X 10 ⁵ /ml。

(2) 500ul of medium containing 10% fetal bovine serum was added to the 24-well plate lower chamber. 200 μl of the cell suspension was added to the chamber and the underlying culture medium were carefully bubble free.

(3) Culturing for 16-24h.

(4) The medium in the chamber was aspirated, the chamber cells were fixed with absolute methanol for 15min and rinsed with PBS.

(5) Seven colors: gu Msa staining solution was added to the 24-well plate, and the membrane was immersed in the staining solution for 20min and washed with PBS.

(6) The inner cells of the membrane were carefully wiped off with a cotton swab and the membrane was air dried.

(7) Photographs were taken under a microscope, 5 high power field counts were taken, and an average was taken.

2 experimental results

Both APY29 and BI-D1870 inhibited proliferation of ACHN cells compared to the blank, and APY29 was more inhibited at the same drug concentration. As can be seen from fig. 3, APY29 can significantly inhibit the proliferative activity of ACHN tumor cells. As can be seen from FIG. 4, the IC50 of APY29 was 0.62. Mu.M. From fig. 5, APY29 can inhibit RCC invasion and migration in vitro. As can be seen from FIG. 6, the inhibition of APY29 was reduced after the RSK4 was knocked down.

Example 4

APY29 inhibits the growth of kidney cancer cells in vivo

1. Experimental method

Nude mice nodulation experiment:

(1) Female BALB/c nude mice of 4-6 weeks old were subjected to nude mice tumorigenesis experiments with 5 nude mice per group, each injected subcutaneously with about 1X 10 ⁶ ESCC cells were dissolved in 200. Mu.l of sterile PBS.

(2) When the tumor volume reaches 150mm ³ At this time, the packet is processed differently. APY29 was administered by intraperitoneal injection (drug dissolution concentration: 5mg/ml, solvent: 2% DMSO+2% Tween-80+30% PEG300+ sterile water) at 15mg/kg or 30mg/kg per day, with BI-D1870 as a positive control.

(3) The observation time of the nude mice in the tumor forming experiment lasts for 20 days, and the mice are treated in time when infection and poor conditions occur. Tumor volume measurements were made every 3-5 days during this period.

(4) Mice were sacrificed at the end of the experimental time, subcutaneous tumors were removed, photographed, weighed, and counted.

2. Experimental results

APY29 inhibited tumor growth more strongly than negative control and RSK4 spectral inhibitor BI-D1870, see figure 7.

Example 5

APY29 and sunitinib synergistically treat renal cell carcinoma angiogenesis

1. Experimental method

HUVEC (human umbilical vein endothelial cells) co-cultured with ACHN were stimulated with sunitinib (2 μm) +APY29 (10 μm) respectively, and the effect of different drugs on HUVEC cell tube formation was examined.

Tube forming experiment:

A. the huvec cells are prepared for digestion after reaching 80% confluence, the cell state after overnight culture is generally better, the cells are collected for counting, and the concentration of the cells is adjusted by gradient dilution according to the requirement, so that the cell state is ensured to be perfect.

B. Adding 200ul HUVEC cell suspension into 24 hole plate, with concentration of about 12-20 ten thousand cells, and making compound hole; after the addition of the cells, the culture plate should not be moved, and the culture plate is placed in a incubator at 37 ℃ for about 4 hours, so that whether blood vessels are formed or not can be observed.

C. Typically, after 15 hours, tube formation will begin to slowly collapse to apoptosis.

D. And (5) result analysis and picture acquisition. After fixation, fluorescent staining can be performed as required.

2. Experimental results

As can be seen from fig. 8-9, the combination significantly reduced abnormally active angiogenesis caused by renal transparent cancer. It is shown that sunitinib and APY29 can play a synergistic role, and the result provides a new idea for treating kidney cancer.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. Use of APY29 in the manufacture of a medicament for the treatment of renal cancer.

2. The use according to claim 1, wherein the renal cancer is renal clear cell carcinoma.

3. The use according to claim 2, wherein said apt 29 is used for the preparation of proliferation inhibitors of renal clear cell carcinoma.

4. The use according to claim 2, wherein said apt 29 is used for the preparation of a blocker of renal clear cell carcinoma invasion and migration.

5. A medicament for the treatment/prophylaxis of diseases associated with RSK4 kinase, which comprises APY29 and sunitinib.

6. Use of the medicament of claim 5 for the preparation of a medicament for inhibiting abnormal active angiogenesis caused by renal clear cell carcinoma.