CN115804844A - Application of inhibitor for targeted inhibition of PAK4 in preparation of tumor prevention and treatment medicament - Google Patents
Application of inhibitor for targeted inhibition of PAK4 in preparation of tumor prevention and treatment medicament Download PDFInfo
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- CN115804844A CN115804844A CN202211172795.5A CN202211172795A CN115804844A CN 115804844 A CN115804844 A CN 115804844A CN 202211172795 A CN202211172795 A CN 202211172795A CN 115804844 A CN115804844 A CN 115804844A
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Abstract
The invention belongs to the technical field of tumor prevention and treatment, and particularly relates to an application of a small-molecule inhibitor for targeted inhibition of PAK4 in preparation of a tumor prevention and treatment medicament. The inhibitor for targeted inhibition of PAK4 is a small molecule inhibitor HTS08604 capable of targeted inhibition of PAK 4. In the application, PAK4 is taken as a target, and the inventor screens and obtains a potential small molecule inhibitor HTS08604 targeting PAK4 by means of computer simulation screening. On the basis, taking prevention and treatment of esophageal squamous cell carcinoma as an example, the inventor preliminarily verifies the practical application effect of the small molecule inhibitor. Experimental results show that the small molecule inhibitor has high safety, presents concentration and time dependence on the proliferation capacity of esophageal squamous cell carcinoma cells, can inhibit migration and invasion of the esophageal squamous cell carcinoma cells, can inhibit metastasis of the esophageal squamous cell carcinoma cells, and shows good application effect.
Description
Technical Field
The invention belongs to the technical field of tumor prevention and treatment, and particularly relates to an application of a small molecular inhibitor for targeted inhibition of PAK4 in preparation of a tumor prevention and treatment medicament.
Background
Esophageal cancer belongs to a common malignant tumor in digestive tract diseases, is influenced by factors such as diseased parts and treatment means, has a poor prognosis effect and a five-year survival rate of only 15-25 percent. Clinically, the cancer is classified into Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC) according to the type of Esophageal cancer diseased tissue.
Surgical treatment is one of the common and most main treatment means of esophageal cancer, and for early patients, better treatment effect can be achieved through the surgical treatment means. For middle and late stage esophageal cancer patients, the survival of the patients is improved by a comprehensive treatment mode of combining preoperative radiotherapy with operation or radiotherapy and chemotherapy. However, esophageal squamous carcinoma is less sensitive to chemotherapeutic drugs than most solid tumors, resulting in a poor overall prognosis. With the development of molecular targeted therapy technology, the molecular mechanism and potential molecular targets for the occurrence and development of esophageal squamous cell carcinoma are explored, which has very important theoretical and technical significance for developing new drugs and improving the prognosis effect of esophageal squamous cell carcinoma.
As a main downstream effector of Rho family small GTPases, PAKs play an important role in cytoskeleton remodeling, cell movement, apoptosis regulation and the like. Members of PAK can be classified into type I (PAK 1-3) and type II (PAK 4-6) according to protein structure and function. Among the PAKs type II, PAK4 is the most studied member and has been shown to be involved in developmental activities such as cytoskeletal remodeling and neural development. It has been shown that the PAK4 gene is located in the human chromosome 19 band 19q13.2 and encodes a protein of approximately 90 kDa in size. Like other PAK isoforms, PAK4 contains a p21-GTPase binding domain (PBD) at the N-terminus and a highly conserved serine/threonine kinase domain at the C-terminus. PAK4 mRNA and protein are ubiquitously expressed in most tissues. The expression level is obviously higher in the embryonic development process, and is lower in adult tissues. Meanwhile, the self-renewal capacity of the neural progenitor cells is also dependent on the expression of PAK 4. The research suggests that besides the regulation of cytoskeleton formation, PAK4 is also involved in basic cell development processes including growth factor pathways, cell proliferation, transcriptional regulation, apoptosis/survival signals and the like, and the participation of these physiological activities provides a new technical path for tumor prevention and treatment, namely: inhibition of PAK4 may result in significant anti-tumor activity.
It has been shown that amplification/mutation or up-regulation of the PAK4 genome in adult tissues has been found in various cancers, such as: breast cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, stomach cancer, oral squamous cell carcinoma, and the like. Thus, monitoring the expression level of PAK4 in adult tissues may also serve as a biomarker for cancer diagnosis. Partial studies have shown that PAK4 also plays an important role in tumor immunotherapy. For example, deletion of the PAK4 gene increases T cell infiltration and is associated with CD8 in mouse models + The T-cell dependent pattern reversed resistance to PD-1 blockade (Zhang J et al, LCH-7749944, alpha novel and potential p21-activated kinase 4 inhibitor, suspensions promotion and inhibition in human structural cells; cancer Lett, 2012). In addition, CAR-T immunotherapy targeting PAK4 to re-plan the vascular microenvironment may improve glioblastoma (Ryu B J et al, discovery and the structural basis of a novel p21-activated kinase 4 inhibitor, cancer Lett, 2014). In summary, PAK4 has been shown to be associated with uncontrolled increased metastasis of tumor cells, cell proliferation, alterations in cell signaling pathways, drug resistance, and modulation of immunotherapy, which makes PAK 4a promising therapeutic target.
Disclosure of Invention
Based on the screening result of the small-molecule inhibitor for targeted inhibition of PAK4, the application aims to provide the small-molecule inhibitor HTS08604 for targeted inhibition of PAK4, and the small-molecule inhibitor is verified by combining the related cell experiments and animal experiments, so that a certain technical basis is laid for prevention and treatment of related tumors (cancers).
The technical scheme adopted by the application is detailed as follows.
The application of an inhibitor for targeted inhibition of PAK4 in preparing a tumor prevention and treatment medicament, wherein the inhibitor for targeted inhibition of PAK4 is a small molecule inhibitor HTS08604 (CAS: 709632-12-2) capable of targeted inhibition of PAK 4; such tumors are in particular, for example, esophageal cancer (more particularly esophageal squamous carcinoma);
the small molecule inhibitor HTS08604 can inhibit PAK4 in a targeted manner, inhibit the EMT progress mode by blocking an ERK/AKT signal channel and play a role in inhibiting the proliferation and migration of esophageal squamous cell carcinoma cells and the invasion of cancer cells by combining a concentration and time-dependent mode; the structural formula of the small molecule inhibitor HTS08604 is as follows:
PAK4 is used as a member of a serine/threonine kinase family, participates in a plurality of signal paths of tumor occurrence and development, has important influence on the physiological functions of tumor growth, proliferation, migration, invasion and the like, and becomes an important target for research and development of antitumor drugs. In the prior art, inhibitors developed for PAK4 can be classified into four types according to their mechanisms of action: the first is an ATP competitive inhibitor, which targets the highly conserved ATP binding pocket of the kinase domain; typical examples are PF-3758309 compounds from the company Peucervi; the second is an allosteric inhibitor, the non-catalytic site of the enzyme molecule is reversibly non-covalently bound to some compound (inhibitor) and undergoes conformational change, thereby changing the enzymatic activity state; the third is miRNAs inhibitor, microRNAs (miRNAs) are used as endogenous non-coding small RNAs, and interfere the translation of mRNAs in a base pairing mode to further regulate gene expression, so that the aim of reducing enzyme activity is finally achieved; the fourth is a naturally derived inhibitor.
Although many studies have been made on inhibitors against PAK4, there are few inhibitor products that have application prospects and can enter clinical stages due to reasons such as bioavailability or use modes, and therefore, screening and development of new PAK4 inhibitors are still urgent. In the research process, the inventor firstly preliminarily verifies that PAK4 is closely related to the occurrence and development of esophageal squamous cell carcinoma, the high expression of PAK4 can promote the proliferation, migration and invasion of esophageal squamous cell carcinoma cells, and meanwhile, PAK4 promotes the proliferation, migration and invasion of esophageal squamous cell carcinoma cells by participating in the ERK and AKT signal pathways and EMT development. This makes PAK 4a better therapeutic target in prevention and treatment of esophageal squamous cell carcinoma.
In the application, PAK4 is taken as a target, and the inventor screens and obtains a potential small molecule inhibitor HTS08604 targeting PAK4 by means of computer simulation screening. On the basis, the inventor preliminarily verifies the practical application effect of the small molecule inhibitor by taking the prevention and treatment of esophageal squamous cell carcinoma as an example. Cell experiment results show that the small molecule inhibitor has high safety, presents concentration and time dependence on the proliferation capacity of esophageal squamous cell carcinoma cells, and can inhibit migration and invasion of the esophageal squamous cell carcinoma cells. Animal experiment results show that the small molecule inhibitor has no obvious toxic or side effect on mice, can obviously inhibit the growth of esophageal squamous cell carcinoma tumors, can inhibit the metastasis of esophageal squamous cell carcinoma cells, and shows a good application effect. The experimental results show that the screened small molecule inhibitor HTS08604 not only has good application potential for preventing and treating esophageal squamous cell carcinoma, but also provides good reference and application basis for preventing and treating other tumors (cancers).
Drawings
FIG. 1 shows that PAK4 is highly expressed in esophageal squamous carcinoma tissues and cells: wherein: (A) Analyzing the expression level of PAK4 in various cancers by the TIMER database; (B) Detecting the expression condition of PAK4 in esophageal squamous carcinoma tissues through an IHC experiment; (C) Statistics of expression differences of PAK4 in esophageal squamous cell carcinoma tissues and paracarcinoma tissues; (D) Westernblot detects the expression levels of PAK4 and p-PAK4 in esophageal squamous carcinoma cells and normal esophageal epithelial cells; (E) Carrying out grey value statistics on the expression difference of p-PAK4 in an esophageal squamous carcinoma cell line and normal esophageal epithelial cells;
FIG. 2 is a schematic view showing the distribution of esophageal squamous carcinoma tissue core carcinoma and tissues beside the carcinoma;
FIG. 3 shows that small molecule inhibitor HTS08604 significantly inhibits the activity of PAK4 protein kinase;
FIG. 4 shows the in vitro and in vivo binding of small molecule inhibitor HTS08604 to PAK4 protein; wherein: (A) The binding condition of a small molecule inhibitor HTS08604 and a recombinant protein PAK 4; (B) The condition that small molecule inhibitor HTS08604 is combined with PAK4 protein in KYSE150 cells; (C) The condition that small molecule inhibitor HTS08604 is combined with PAK4 protein in KYSE450 cells;
FIG. 5 is a graph showing the binding pattern of HTS08604 to PAK4 protein; simulating the binding site of HTS08604 and PAK4 by using a computer molecular docking model;
FIG. 6 shows that HTS08604 competes with ATP for binding to PAK4 protein kinase;
FIG. 7 is a graph showing the toxic effect of HTS08604 on normal esophageal epithelial cells and esophageal squamous carcinoma cells; wherein: (a) HTS08604 is non-toxic to normal esophageal epithelial cells; (B) Toxic effects of HTS08604 on esophageal squamous carcinoma cells KYSE 150; (C) The toxic effect of HTS08604 on esophageal squamous carcinoma cells KYSE 450;
FIG. 8 shows that HTS08604 inhibits the proliferation of esophageal squamous carcinoma cells; wherein: (A-B) HTS08604 inhibited proliferation of esophageal squamous carcinoma cells KYSE150 (A) and KYSE450 (B) in vitro;
FIG. 9 shows that HTS08604 has an inhibitory effect on the clonogenic capacity of KYSE150 and KYSE450 cells of esophageal squamous carcinoma;
FIG. 10 shows HTS08604 inhibiting the migration and invasion of esophageal squamous carcinoma cells; wherein: (A-B) scratch test to detect the influence of different concentrations of HTS08604 on the migration capability of the esophageal squamous carcinoma cell lines KYSE150 (A) and KYSE450 (B); (C-D) Transwell experiment to examine the effect of different concentrations of HTS08604 on the migration and invasion capabilities of esophageal squamous cell carcinoma cell lines KYSE150 (C) and KYSE450 (D);
FIG. 11 is a graph showing the level of phosphorylation of HTS08604 inhibiting the downstream pathway of PAK4 protein;
FIG. 12 is a graph showing that HTS08604 inhibits EMT progression in esophageal squamous carcinoma cells (KYSE 150, KYSE 450);
FIG. 13 shows that the expression level of PAK4 protein is reduced in esophageal squamous carcinoma cell lines KYSE150 and KYSE 450;
FIG. 14 is a drawing showing that HTS08604 targeting PAK4 inhibits the proliferation of esophageal squamous carcinoma cells; wherein: (A-B) cell proliferation experiment is used for detecting the influence of HTS08604 on the proliferation capacity of KYSE150 (A) and KYSE450 (B) of esophageal squamous carcinoma cell lines for knocking-down PAK 4; (C-D) plate cloning experiment to detect the influence of HTS08604 on clone forming capability of the esophageal squamous carcinoma cell line KYSE150 (C) and KYSE450 (D) for knocking-down PAK 4;
FIG. 15 is a graph showing that HTS08604 targeting PAK4 inhibits the migration and invasion of esophageal squamous carcinoma cells; wherein: (A-B) the Transwell experiment detects the influence of HTS08604 on the migration and invasion of the esophageal squamous carcinoma cell lines KYSE150 (A) and KYSE450 (B) for knocking-down PAK 4;
fig. 16 is HTS08604 has no effect on mouse body weight and gut; wherein: (a) HTS08604 had no effect on the body weight of SCID mice; (B) HE staining detection HTS08604 has no damage to mouse viscera;
FIG. 17 shows that HTS08604 inhibits esophageal squamous cell proliferation in vivo; wherein: (A) pictures of tumors in each group of mice; (B) statistics of tumor volumes for each group; (C) Counting the weight of each group of tumors and calculating the tumor inhibition rate;
FIG. 18 is a record of the body weight of mice in each group;
FIG. 19 is a graph showing that HTS08604 down-regulates the phosphorylation levels of ERK and AKT in vivo;
FIG. 20 is a graph showing that HTS08604 inhibits the expression of EMT-associated markers in CDX tissue;
FIG. 21 is a diagram of HTS08604 regulating expression of a marker of interest in CDX tissue; wherein: (A) HTS08604 regulates the phosphorylation levels of ERK, AKT in CDX tissue; (B) HTS08604 regulates the expression of EMT-associated marker proteins in CDX tissue;
FIG. 22 HTS08604 inhibits esophageal squamous cell metastasis in vivo; wherein: (A) Bioluminescence imaging of mice treated with HTS08604 (35 mg/kg and 70 mg/kg) by tail vein injection of KYSE150-Luc cells; (B) carrying out statistical analysis on fluorescence intensity of mouse lung; (C) HE staining showed lung metastasis;
in the image-related data processing: * p <0.05, p <0.001.
Detailed Description
Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
Biological material: human esophageal squamous carcinoma cell lines (KYSE 150, KYSE30, KYSE450 and KYSE 510), human renal epithelial 293T cells (HEK 293T) and esophageal epithelial cells (HET-1A) are all cell lines (systems) commonly used in pathophysiology research, can be obtained from public channels, and are stored for a long time as relevant research materials in colleges and universities; CB17/SCID mice (SPF grade), female mice, 4-5 weeks old mice, body weight of about 15-20g, purchased from Beijing Wintonli laboratory animal technology, inc.; feeding culture conditions: the temperature is 18-22 ℃, the humidity is 60-70%, and the feed and the water are fed after being sterilized.
The main reagents are as follows: HTS08604, available from shanghai pottery biotechnology limited; PF-3758309 (ATP competitive p21-activated kinase (PAK) inhibitor) available from poisonous Scissors of Shanghai Ruichi, inc.; FBS (fetal bovine serum), DMEM medium, RPMI-1640 medium, product of BIOLOGICALINDUSTRIES; g418, puromycin, penicillin (100X), BCA protein concentration determination kit, beijing Sorbao science and technology company; jetPRIME transfection reagent, product of Polyplus, france; polybrene, product of Sigma company; 0.25% trypsin, ultra-sensitive ECL chemiluminescence kit, shanghai Bin Yuntian Biotech limited product; PAK4 (active), signalChem product; kinase Buffer (10X), ATP, cell Signaling Technology, USA product; CCK8 kit, shanghai pottery biotechnology limited; matrigel, product of corning corporation, usa; sheep/rabbit SP kit, rabbit/mouse SP kit, DAB color development kit, goat anti-rabbitIgG-HRP, goatanti-mouse IgG-HRP, products of Beijing China fir Jinqiao biotechnology, inc.; PAK4 antibody, p-PAK4 (Ser 474) antibody, AKT antibody, p-AKT (Ser 473) antibody, ERK1/2 antibody, p-ERK1/2 (Thr 202/Tyr 204) antibody, E-cadherin antibody, N-cadherin antibody, beta-catenin antibody, p-beta-catenin (Ser 675) antibody, a product of Cell Signaling Technology company; vimentin antibody, available from Affinity Biosciences; p-Serine antibody, product of Abcam corporation; GAPDH antibody, from Hangzhouxian to Biotechnology GmbH.
Used in the experimental procedure: complete RPMI-1640/DMEM medium (500 ml, RPMI-1640/DMEM medium 450 ml, fetal bovine serum (10% FBS) 50 ml, streptomycin (100X) 5 ml), G418 (100 mg/ml: G418, 1G, 1M HEPES), 4% paraformaldehyde, 0.1% crystal violet, 33% glacial acetic acid, RIPA cell lysate (RIPA, 500. Mu.l, PMSF (100 mM), 5. Mu.l), 4 XSTris-HCl/SDS (pH 6.8), 4 XSTris-HCl/SDS (pH 8.8), 10% ammonium persulfate, 10% separation gel, 8% separation gel, 10 XTTBbuffer, 10 XTBS buffer, 5% skim milk-ST, 5% Tris-ST, 5 XTRIP buffer, loading buffer, 0.1M Tris-HCl buffer, tris-HCl + NaCl buffer (pH 8.5), and antibody dilution, etc., and can be prepared or operated conventionally.
The main apparatus is as follows: a full wavelength microplate reader, thermo corporation, usa; protein transilluminator, horizontal electrophoresis apparatus, BIO-RAD, USA; ultrasensitive full-automatic imaging analysis systems, protein Simple, usa; small animal in vivo imager, perkinElmer, usa; panoramic tissue cell quantitative scanning and analysis system, austria pharmaceutical science and technology company.
Example 1
The prior art shows that PAK4 is closely related to the occurrence and development of tumors, therefore, based on the prevention and treatment needs of esophageal squamous cell carcinoma, the inventor firstly carries out research and analysis on the expression conditions of PAK4 in esophageal squamous cell carcinoma and normal epithelial tissues of esophagus, and the process is briefly described as follows.
Based on the TIMER database, the inventor firstly studies and analyzes the expression level of PAK4 in esophageal squamous carcinoma and normal esophageal epithelium, and the result shows that PAK4 is highly expressed in various cancers (the result is shown in figure 1A). Among them, PAK4 expression has significant difference in ESCC (esophageal squamous cell carcinoma) and normal esophageal epithelium. The method lays a preliminary research foundation for preventing and treating esophageal squamous cell carcinoma by taking PAK4 as a target.
To further verify whether PAK4 is highly expressed in ESCC tissues compared to paracancerous tissues, the inventors further performed research and analysis using an ESCC tissue chip (purchased from shanghai core supermicro, which contains 103 ESCC tissues and 77 paracancerous tissues, as shown in fig. 2). In combination with immunohistochemical staining analysis results (fig. 1B, fig. 1C), it can be seen that: PAK4 is highly expressed in ESCC tissues compared to paracarcinoma tissues. Further Western blot experiments showed that PAK4 is highly expressed in esophageal squamous cell carcinoma cell lines compared to normal esophageal epithelial cells (fig. 1D, fig. 1E). Meanwhile, the expressions of PAK4 and p-PAK4 in esophageal squamous carcinoma cell lines KYSE150 and KYSE450 are both high (the two cell lines are selected for verification in a subsequent in vitro cell test).
It should be explained that, in immunohistochemical staining, reference is made to the existing routine procedures, or specific reference may be made to the following:
(1) Tissue embedding and sectioning: placing the tissue (which is soaked in neutral formaldehyde in advance for fixing) in an embedding box, marking, and dehydrating for 12 h overnight; then, taking out the dehydrated tissue, wrapping the dehydrated tissue with a wax block melted in advance, placing the wrapped tissue on ice for solidification and demolding, and finally taking out the wax-wrapped tissue; cutting off excessive wax on the surface of the tissue to fully expose the tissue, slicing by a slicer (the thickness is 2 μm), then placing the slice in a 42 ℃ water bath to expand the tissue, and fishing out and drying water; (2) dewaxing and hydrating the section tissues: baking the glass slide with the tissue section in a 65 ℃ oven until the wax block on the glass slide is completely melted; the slices were then sequentially treated to dewax hydration in the following reagents: 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol, standing for 5min respectively; finally cleaning with 1 × TBST for 5min for 3 times; (3) repair and sealing: placing the processed slices (which can be placed on a slide rack) in a repairing liquid (the liquid level is required to completely cover the slices), placing in a microwave oven, heating until the repairing liquid is boiled, keeping boiling for 10min, and cooling at room temperature for 2h; subsequently, washing with 1 × TBST for 5min 3 times; wiping water on the edge of the slide tissue with absorbent paper, dripping hydrogen peroxide solution to cover the tissue, and storing in dark for 15min; cleaning with 1 × TBST for 3 times, each time for 5min; wiping water on the edge of the slide tissue with absorbent paper, dripping sealing liquid, and storing in dark for 15min to complete sealing; (4) antibody binding: dropping primary antibody (diluted with 1% BSA-TBST in advance) on the slide tissue after the above blocking is completed, and covering with a sealing film (to keep the tissue wet), followed by placing the slide in a wet box and incubating overnight at 4 ℃; after the incubation is finished, washing for 5min with 1 × TBST for 3 times; wiping water on the edge of the slide tissue with absorbent paper, dripping a secondary antibody corresponding to the primary antibody on the slide tissue, then placing the slide tissue in an oven at 37 ℃ for incubation for 15min, and cleaning with 1 × TBST for 3 times, 5min each time; wiping water on the edge of the slide tissue with absorbent paper, dripping horse radish peroxidase marker on the tissue, and storing in dark for 15min; finally cleaning with 1 × TBST for 5min for 3 times; (5) staining, enveloping and scanning analysis: preparing DAB working solution according to the reference instruction, and performing DAB staining on the slide tissue after the antibody is combined (stopping staining by tap water when the staining is finished); subsequently, hematoxylin counterstaining was performed: soaking in hematoxylin for 3 min, and washing with tap water for 5min; differentiating with 1% hydrochloric acid for 1 s, and washing with tap water for 15min; reference is again made to the following sequential gradient treatment: soaking 50% ethanol, 70% ethanol, 90% ethanol, 100% xylene, and 100% xylene in each solution for 5min; then, dripping neutral resin on the slide glass tissues after the hematoxylin counterstaining, and covering a cover glass for mounting; finally, the panoramic scanner scans and analyzes the positive rate (using HistoQuest software).
Example 2
On the basis of the embodiment 1, in view of the fact that PAK4 can be used as a potential target for prevention and treatment of esophageal squamous cell carcinoma, and the technical advantages and potential application prospects of the small-molecule inhibitor are considered, the inventor further screens the specific affinity PAK4 protein kinase small-molecule inhibitor by a virtual screening method based on a structure. The specific process is briefly described as follows.
Firstly, downloading a crystal structure of PAK4 from a protein structure database (www.rcsb.org), and constructing a binding conformation of the PAK4 and a small molecule compound with known activity by a molecular docking method; subsequently, through the molecular dynamics simulation of the complex structure of the PAK4 and the known small molecule compound, the structural change condition of the PAK4 and small molecule compound structure in the simulation process is observed; finally, performing clustering analysis on the structure in the simulation track, and selecting a representative PAK4 structure from each type of conformation; and carrying out virtual screening based on the structure on the selected PAK4 structure and the small molecule library, and screening out a potential PAK4 small molecule compound inhibitor according to the binding free energy obtained by a scoring function and the binding mode of the small molecule and the PAK 4.
And finally, selecting 12 small-molecule compounds with the highest score and higher affinity with the crystal structure of the PAK4 protein as candidate small-molecule inhibitors (the 12 compounds contain HTS08604 of the application, and the other 11 compounds are not related to the application and are not described in detail).
On the basis of the screening result, in order to further verify whether the candidate small molecule compound obtained by screening has an inhibitory effect on the activity of PAK4 kinase, the inventors further experimentally verified the binding condition of the small molecule inhibitor HTS08604 and PAK4 kinase by using in vitro kinase experiments, protein pull-down experiments and the like, and the specific experimental conditions are briefly described as follows.
In vitro kinase experiment verification
First, the experimental design and the first part of the reaction system are designed as follows:
adding the above reagents into 8 EP tubes of 200 μ l, respectively, mixing, incubating at room temperature for 15min, and keeping out of the sun; A. b, C and D are four experimental groups; subsequently, a second part of the reaction system was formulated as follows:
adding the second part of reaction system into the corresponding EP tube of the first part of reaction system, uniformly mixing, and incubating in a water bath kettle at 30 ℃ for 30 min; after the incubation is finished, 5 mul of 6 Xloading is added into each tube to terminate the reaction; and finally, boiling the sample in a water bath kettle at 100 ℃ for 5min, standing on ice for 2 min, performing instantaneous centrifugation, then loading the sample, and performing detection analysis by referring to SDS-PAGE electrophoresis in a Western blot experiment. A. In four experimental groups, the final concentrations of the small molecule inhibitor HTS08604 (prepared by dissolving the small molecule inhibitor HTS08604 in DMSO solution in advance) are: 1.μ M, 5 μ M, 10 μ M, 20 μ M. In the experimental design, in-vitro kinase experiments are carried out by using recombinant PAK4 protein with kinase activity and inactive beta-catenin, the beta-catenin is used as a kinase substrate, p-beta-catenin (Ser 675) is used as a detection index, and a PAK4 small-molecule inhibitor PF-3758309 is used as a positive control (the concentration is 10 mu M).
The results of the experiment are shown in FIG. 3. Analysis can see that: HTS08604 has strong inhibiting effect on PAK4 protein kinase activity. Specifically, the method comprises the following steps: HTS08604 was able to inhibit the phosphorylation level of β -catenin at Ser675 site and in a dose-dependent manner. This result suggests that the small molecule inhibitor HTS08604 may directly bind to and inhibit PAK4 protein kinase activity.
(II) protein pull-down experiment verification
To test whether HTS08604 exerts its relevant functional effect by binding to PAK4 protein during inhibition of PAK4 protein kinase activity, the inventors performed further protein pull-down experiments. The specific procedures and results are summarized below.
(1) Coupling of small molecule compound HTS08604 with Sepharose 4B
First, 0.3 g of Sepharose 4B (hydrogen bromide activated) was weighed into two 50 ml centrifuge tubes (wrapped with tinfoil paper for storage in the dark); subsequently, 30 ml of 1 mM HCl was added to activate Sepharose 4B per tube, after slowly rotating for 5min at room temperature, centrifuging for 2 min at 2000 rpm and standing for 5min, removing the supernatant with a vacuum pump and retaining 5ml of supernatant (to avoid loss of Sepharose 4B), this step being repeated three times (to ensure adequate activation of Sepharose 4B); then, taking two new 15 ml centrifuge tubes (coated with tin foil paper to avoid light) as a control group and an experimental group respectively, transferring the activated Sepharose 4B into the two new centrifuge tubes, and adding 5ml of coupling buffer solution respectively; the experimental group was added with 3 mg of small molecule compound HTS08604 (previously dissolved in DMSO), and the control group was added with an equal amount of DMSO; after the addition was complete, the tube was gently mixed by turning upside down and rotated overnight at 4 ℃ (for primary coupling); centrifuging the tube at 2000 rpm for 2 min, standing for 5min, and removing supernatant with vacuum pump; adding 5ml of coupling buffer solution into each of the two centrifuge tubes, rotating at low speed for 5min, centrifuging at 2000 rpm for 2 min, standing for 5min, and removing supernatant by using a vacuum pump (repeating the steps of adding the coupling solution → centrifuging → extracting the supernatant for 3 times); finally 5ml of 0.1M Tris-HCl (pH 8.0) solution was added to each tube and spun overnight at 4 ℃; on the third day, centrifuging the tube at 2000 rpm for 2 min, standing for 5min, and removing the supernatant by a vacuum pump; adding 5ml of 0.1M acetic acid + 0.5M sodium chloride solution (pH 4.0) into each tube, rotating at low speed for 5min at room temperature, centrifuging at 2000 rpm for 2 min, standing for 5min, and removing supernatant with vacuum pump; adding "0.1M Tris-HCl + 0.5M sodium chloride solution" (pH 8.5), rotating at low speed for 5min at room temperature, centrifuging at 2000 rpm for 2 min, and standing for 5min, and then removing the supernatant by a vacuum pump (the steps of adding "0.1M Tris-HCl + 0.5M sodium chloride solution" → centrifugation → removing the supernatant are repeated 3 times); then adding 10 ml of 1 XPBS into each tube for washing, rotating at low speed for 5min at room temperature, centrifuging at 2000 rpm for 2 min, standing for 5min, and then pumping out supernate by using a vacuum pump; finally, 1 ml of 1 XPBS was added to each tube and stored at 4 ℃.
(2) PAK4 protein pull-down validation
Firstly, two 1.5ml EP tubes are prepared, 100 μ l of beads (namely Sepharose 4B after coupling) of corresponding groups (a control group and an experimental group) are respectively added, 500 μ g of cell lysate containing esophageal squamous carcinoma cells KYSE450 or KYSE150 (the cell lysate is used for carrying out lysis treatment on the esophageal squamous carcinoma cells KYSE450 and KYSE150 in advance and quantifying protein), 1 × NETEW buffer solution is used for quantifying to 500 μ l, and the shaking table is rotated at 4 ℃ for incubation for 36 h; in the period, an Input sample is prepared by referring to a method for preparing a protein sample in the western blot, the loading protein amount is 20 mu g, and the sample is stored at the temperature of-20 ℃ for later use after the preparation is finished; after the incubation is finished, centrifuging the EP tube at 4 ℃ and 5000 rpm for 3 min, and pumping out the supernatant by a vacuum pump; adding 500 μ l of 1 × NETEW buffer solution into each tube, performing rotary incubation for 5min at 4 deg.C with a shaking table, centrifuging for 3 min at 4 deg.C and 5000 rpm, removing supernatant with a vacuum pump, and repeating for 3 times (i.e., adding 1 × NETEW buffer solution → incubating → centrifuging → extracting supernatant for 3 times, and finally performing the final evacuation to the greatest extent); then, after estimating the volume occupied by the residual beads in the EP tube, adding a proper amount of 6 × loading (about 45 μ l is added in the application) into each tube, boiling the sample at 100 ℃ for 5min, placing the sample on ice for 2 min, extracting the supernatant and then loading the sample; finally, according to the following steps: and sequentially loading the Input, the DMSO-Sepharose 4B and the HTS 08604-Sepharose 4B, and detecting and analyzing SDS-PAGE electrophoresis in Western blot.
The results of the experiment are shown in FIG. 4. After analysis, it can be seen that: recombinant PAK4 protein with kinase activity could bind to HTS 08604-conjugated Sepharose 4B but not to DMSO-conjugated Sepharose 4B (negative control) (fig. 4A). While the results of the pull-down experiment using KYSE150 and KYSE450 cell lysates show that: HTS08604 can bind to PAK4 protein (fig. 4B, fig. 4C).
Based on the above results, in combination with the PAK4 crystal structure, the inventors further constructed a molecular docking model of HTS08604 and PAK4 by means of molecular docking software simulation, and simulated the specific site where PAK4 binds to HTS08604. The results are shown in FIG. 5. It can be seen that: HTS08604 was able to bind to PAK4 protein at ILE-327, MET-395, PHE-397, LEU-398, and LEU-447 sites.
(III) competitive ATP binding assay
Based on the above experimental results, to further verify whether the binding of the small molecule compound HTS08604 to PAK4 protein kinase is related to ATP. The inventor utilizes active PAK4 protein kinase, sepharose 4B coupled with small molecular compound HTS08604 and ATP with different concentrations to establish an in vitro reaction system, and utilizes Western blot experiment to detect the expression quantity of the pulled-down PAK4 protein. The specific experimental procedures are summarized below.
First, the reaction system was prepared in accordance with the following table, each tube (EP tube) system was 950. Mu.l (ATP concentration in the table is the final concentration, i.e., ATP final concentration is 0, 10, 100 and 1000. Mu.M, respectively)
After the preparation is finished, incubating overnight at 4 ℃ on a shaking table; after the incubation is finished, 50 μ l of coupled small molecule compound HTS08604 (refer to the above operation) is added to each tube, the final volume of each tube is 1 ml, and the tube is incubated for 2h at 4 ℃ in a rotating way; after the incubation is finished, centrifuging at 4 ℃ and 12000 rpm for 90 s, and extracting a supernatant; after estimating the volume of the beads (about 30. Mu.l) after centrifugation, 6. Mu.l of 6 × loading buffer is added into each tube and mixed evenly, the sample is boiled at 100 ℃ for 5min, placed on ice for 2 min, and loaded after instantaneous separation; reference to the SDS-PAGE electrophoretic procedure in the western immunoblotting procedure, as follows: protein marker, 0mM, 1 mM, 10 mM, 100 mM ATP concentration were loaded sequentially. The gray scale value of the strip is calculated by image J software according to the result after exposure.
The results are shown in FIG. 6. Analysis can see that: with the increasing concentration of ATP, the binding capacity of the small molecule compound HTS08604 and the PAK4 protein kinase is gradually reduced, which indicates that the binding capacity of the HTS08604 and the PAK4 protein kinase depends on the concentration of ATP and inhibits the activity of the PAK4 protein kinase in an ATP competition mode.
In the introduction of the above experiment, the following can be referred to for the operation of Western blotting (Western blot):
(1) Collecting cells: after the cultured cells are cleaned, the cells are collected into a 1.5ml EP tube, centrifuged at 5000 rpm for 5min at 4 ℃, and the supernatant is discarded; (2) cell lysis: adding a proper amount of cell lysate (generally 100 to 200 mu l) according to the cell precipitation amount, uniformly mixing, placing on ice for 30 min, and performing vortex oscillation once every 10 min; centrifuging at 12000 rpm for 30 min at 4 ℃ after the cracking is finished; after the centrifugation is finished, extracting the supernatant of the middle layer (avoiding sucking the surface membrane and the cell debris at the bottom) and transferring the supernatant into a new 1.5ml centrifuge tube for later use; (3) BCA method for measuring protein concentration: first, BCA protein standard solutions (PBS dilution: 0 mg/ml,0.025 mg/ml,0.05 mg/ml,0.1 mg/ml,0.2mg/ml,0.3 mg/ml,0.4 mg/ml,0.5 mg/ml) were prepared; subsequently, a 96-well plate is taken, and the diluted gradient BCA protein standard solution and the protein to be tested diluted 10 times (1. Mu.l of protein + 9. Mu.l of PBS) with 1 XPBS are respectively added into the 96-well plate (three multiple wells are set for each sample); then adding 200 mul of BCA working solution into each hole, and after the sample addition is finished, placing the mixture in an incubator at 37 ℃ for incubation for 30 min; after incubation is finished, an enzyme-labeling instrument detects the light absorption value of each hole under the wavelength of 562nm, the BCA concentration is used as a horizontal coordinate, the light absorption value is used as a vertical coordinate, a standard protein curve is drawn, and the concentration of the protein to be detected is calculated and analyzed; (4) SDS-PAGE electrophoresis: preparing a separation glue (lower glue) and a concentrated glue (upper glue) by referring to the prior art; when the sample is loaded, the protein mass added into each hole is 60 mug (the total volume is 60 mug, wherein 6 × loading buffer is 10 mug); during electrophoresis detection, protein molecules run from a negative electrode to a positive electrode, the protein molecules run to the boundary of concentrated gel and separation gel at a constant voltage of 90V, the voltage is adjusted to be 120V, and the electrophoresis time is determined according to the position of a target protein (the target protein is stopped after separation); after electrophoresis, performing membrane conversion treatment (PVDF membrane, constant-pressure membrane conversion, 90V and 2 h); after the membrane transfer is finished, blocking nonspecific protein binding sites in the PVDF membrane by using 5% skim milk-TBST (incubating for 90min at room temperature in a shaking table); after the sealing is finished, sequentially carrying out primary antibody treatment and secondary antibody treatment by referring to the specification; after the secondary antibody incubation was completed, the membrane was washed with 1 × TBST (5 min/time, 3 times). According to the liquid A: solution B =1, ECL luminescent solution was prepared at a ratio of 1, background exposure was adjusted, and Image J was analyzed for gray value.
Example 3
Based on example 2, the inventors further performed in vitro cell experiments to further evaluate the effect of HTS08604 on the growth of esophageal squamous carcinoma cells. The specific experimental procedures and results are summarized below.
Toxic effects of HTS08604 on esophageal squamous carcinoma cells
Taking esophageal squamous carcinoma cells cultured until the cell confluence reaches 80-90%, cleaning with 1 XPBS, adding pancreatin for digestion, and adding a complete culture medium to stop digestion after digestion is completed; 800 Centrifuging at rpm for 5min, removing supernatant, adding complete culture medium to resuspend cell precipitate, and gently blowing to obtain single cell suspension; 1X 10 cells according to KYSE150 5 1.0X 10 cells/ml KYSE450 cells 5 Preparing a cell suspension at a concentration of one/ml; the prepared cell suspension was added to a 96-well plate in an amount of 100. Mu.l per well (5 wells per concentration), while a blank well (cell-free medium) and a control well (cell-containing medium) were placed, and the wells were surrounded withEdge sealing with 1 × PBS; putting into an incubator for 18 h; after the cells adhere to the wall, the original culture medium in the 96-well plate is poured off, and 100 mul of complete culture medium containing the medicines is added into each well (corresponding complete culture medium is prepared according to different medicine concentration designs), and the time is recorded as 0 h; at 48h and 72 h, CCK8 dilutions (CCK 8: complete medium =1 9) were added, respectively, and absorbance at 450 nm was measured after incubation in an incubator for 2h; and finally, carrying out statistical analysis on the data and drawing a line graph, wherein the abscissa is the treatment time of the medicine, and the ordinate is the cell survival rate. In the experimental design, 0, 2.5, 5, 10, 20, 40 and 80 μ M HTS08604 acts on HET-1A of normal esophageal epithelial cells and esophageal squamous carcinoma cell lines (KYSE 150 and KYSE 450), respectively, the viability of the cells is detected at 48h and 72 h respectively, the IC50 is calculated, and finally the toxic effect of the small-molecule compound HTS08604 on the HET-1A of normal esophageal epithelial cells and the esophageal squamous carcinoma cell lines (KYSE 150 and KYSE 450) is evaluated.
The results of the experiment are shown in FIG. 7. It can be seen that: 0. 2.5, 5, 10, 20, 40, 80 μ M HTS08604 had no significant toxicity to normal esophageal epithelial cells HET-1A (FIG. 7A), indicating that the small molecule compound HTS08604 has better safety to normal esophageal epithelial cells. The small molecule compound HTS08604 has certain toxicity to esophageal cancer cell lines KYSE150 and KYSE450 (FIGS. 7B and 7C). When HTS08604 acts on esophageal squamous carcinoma cell lines for 48h and 72 h, the IC50 values of KYSE150 for esophageal squamous carcinoma cells are respectively 20.01 mu M and 10.08 mu M, and the IC50 values of KYSE450 for esophageal squamous carcinoma cells are respectively 19.03 mu M and 8.509 mu M. This result indicates that HTS08604 has stronger toxicity to esophageal squamous carcinoma cells than normal esophageal epithelial cells.
(II) Effect of HTS08604 on esophageal squamous carcinoma cell proliferation
With reference to the foregoing experimental procedures, as follows: KYSE150 cells at 3X 10 4 One/ml, 5X 10 cells of KYSE450 4 Preparing a cell suspension at a concentration of one/ml; and carrying out operations such as plate laying, culture, medicine adding, measurement and the like. In experimental design, 0, 1, 5, 10 and 20 mu M of small molecule compound HTS08604 was used to treat esophageal squamous carcinoma cell lines KYSE150 and KYSE450, respectively, and the fine particles were detected at 0, 24, 48, 72 and 96 h, respectivelyAbsorbance of the cell at 450 nm. The results of the experiment are shown in FIG. 8. It can be seen that: HTS08604 has the capacity of inhibiting the proliferation of esophageal squamous carcinoma cells and is dependent on concentration and time.
On the basis of the experimental results, in order to further evaluate the inhibition of HTS08604 on the cloning capacity of esophageal squamous cell carcinoma cells, the inventor uses 0, 1, 5, 10 and 20 mu M of small molecule compound HTS08604 to treat esophageal squamous cell carcinoma cells KYSE150 and KYSE450, changes the dosing culture medium every three days, photographs after two weeks of culture to observe the clone size and counts the clone number. The results of the experiment are shown in FIG. 9. Analysis shows that: the small molecule compound HTS08604 inhibits the clonogenic formation of esophageal squamous carcinoma cells in a dose-dependent manner, the size and number of clones being influenced by this compound. Among them, HTS08604 at 5, 10, and 20 μ M significantly (p < 0.001) inhibited the ability to form clones in both esophageal squamous carcinoma cells, indicating that HTS08604 can indeed inhibit the proliferation of esophageal squamous carcinoma cells.
(III) cell scratch, migration and invasion experiments
Based on the above experiments, in order to further evaluate the effect of HTS08604 on the migration and invasion capacity of esophageal squamous carcinoma cells, the inventors further performed cell scratching and Transwell experiments. The specific experimental procedures and results are summarized below.
(1) Cell scratch test
With reference to the foregoing experimental procedures, as follows: the number of cells was 1.2X 10 6 Preparing a cell suspension at a concentration of one/ml; taking a 6-hole plate (four transverse lines with the interval of 7 mm are drawn at the outer bottom as a mark), adding 2 ml of complete culture medium into each hole, adding 1 ml of the cell suspension into each hole, slightly shaking to uniformly spread cells in the holes, and culturing for 24h in an incubator (ensuring that the cell confluence reaches 100%); drawing three vertical lines in a 6-hole plate by using a 200-microliter gun head at a position vertical to a transverse line drawn on the previous day; after the exfoliated cells are washed away by 1 XPBS, adding 3 ml of medicine-added culture medium (incomplete culture medium) with corresponding concentration into each hole, taking a scribing picture in each hole under a microscope, recording specific positions and time, and taking the scribing picture as 0 h of a scratching experiment; continuously placing the 6-hole plate in an incubator for culturing; according to different cellsThe moving speed of the moving speed is taken after a certain time interval (the moving speed is consistent with the scribing position of 0 h); and finally, comparing the scribing width with the 0 h time point according to different time points, and performing statistical analysis.
(2) Cell migration assay
With reference to the experimental procedures described above, 1.5X 10 inoculations per well were made 4 Cell count, cell concentration 7.5X 10 prepared with incomplete medium 4 Cell suspension per ml; 24-well plates and Transwell chambers (0.8 μ M pore size), 800 μ l complete medium was added to the lower chamber and 200 μ l cell suspension was added to the upper chamber (final concentrations of 0, 1, 5, 10, 20 μ M small molecule compound HTS08604 were added, respectively); culturing for 24h in an incubator; after the culture is finished, taking out the 24-hole plate, discarding the cell suspension in the upper chamber and the complete culture medium in the lower chamber, cleaning the small chamber with 1 XPBS, adding 200 mu l of 4% paraformaldehyde in the upper chamber, adding 800 mu l of 4% paraformaldehyde in the lower chamber, and fixing at room temperature for 30 min; after the fixation is finished, a vacuum pump is used for pumping away paraformaldehyde, the small chamber is cleaned by 1 XPBS, 200 mu l of 0.1% crystal violet is added into the upper chamber, 800 mu l of 0.1% crystal violet is added into the lower chamber, and the 24-hole plate is placed into a 37 ℃ oven for dyeing for 30 min; washing crystal violet after dyeing is finished, washing each small chamber with ultrapure water, taking a picture after drying, and randomly selecting five visual fields under a microscope for taking a picture; finally, the cells in each field were counted using image J software and statistically analyzed.
(3) Cell invasion assay
The matrigel dissolved in advance was prepared according to the following formula: incomplete medium =1, adding 70 μ l of diluted matrigel solution (avoiding air bubbles) to the upper chamber of the chamber, and then placing the 24-well plate in a 37 ℃ incubator to be solidified (generally, 4h of solidification is needed); according to 5X 10 per hole 4 Individual cells were used as standards, and the cell concentration was 2.5X 10 using incomplete medium, as described above 5 Cell suspension of each ml, and adding small molecule compound HTS08604 (0, 1, 5, 10, 20 mu M) with different concentrations into the cell suspension; add 800. Mu.l complete medium into the lower chamber of the chamber and 200. Mu.l cell suspension into the upper chamber; culturing in an incubator for 24 hours; after the culture was completed, the 24-well plate was taken out, and the upper chamber was discardedAfter the cell suspension, the matrigel and the complete culture medium in the lower chamber are washed by 1 XPBS, 200 mul of 4% paraformaldehyde is added into the upper chamber, 800 mul of 4% paraformaldehyde is added into the lower chamber, and the chamber is fixed for 30 min at room temperature; after the fixation is finished, a vacuum pump is used for pumping away paraformaldehyde, the small chamber is cleaned by 1 XPBS, 200 mu l of 0.1% crystal violet is added into the upper chamber, 800 mu l of 0.1% crystal violet is added into the lower chamber, and the 24-hole plate is placed into a 37 ℃ oven for dyeing for 30 min; washing crystal violet after dyeing is finished, washing each small chamber with ultrapure water, airing, taking a picture, and randomly selecting five visual fields under a microscope to take a picture; and finally, counting and carrying out statistical analysis by imageJ software.
In the scratching experiment, the esophageal squamous carcinoma cell lines KYSE150 and KYSE450 are treated with 0, 1, 5, 10 and 20 mu M of small molecule compound HTS08604 respectively, and the migration of the cells at the same position is recorded by photographing after 0, 12 and 24 hours. The results of the experiment are shown in FIG. 10. It can be seen that: compared with a control group, the migration capability of esophageal squamous carcinoma cell lines KYSE150 and KYSE450 is obviously inhibited after 12 h and 24h of treatment of the small-molecule compound HTS08604 (FIG. 10A and FIG. 10B).
The results of cell migration and invasion experiments further using Transwell experiments show that: HTS08604 dose-dependently inhibited esophageal squamous carcinoma cell migration and invasion, with HTS08604 at 10 μ M and HTS at 20 μ M significantly (p < 0.001) inhibiting esophageal squamous carcinoma cell migration and invasion in both esophageal squamous carcinoma cells (fig. 10C, fig. 10D).
(IV) Signal Conditioning Path
The ERK/AKT signal pathway is a classical pathway for regulating cell proliferation, when PAK4 participates in regulation of the ERK/AKT signal pathway, the expression of PAK4 is up-regulated, and the phosphorylation levels of ERK (Extracellular signal-regulated Kinase) and AKT (Protein Kinase B) are increased. To further verify whether small molecule compound HTS08604 regulates cell proliferation via the ERK/AKT signaling pathway, the inventors collected esophageal squamous carcinoma cell lines (KYSE 150, KYSE 450) treated with 0, 1, 5, 10 and 20 μ M HTS08604 for 24h and examined phosphorylation levels of ERK, AKT and PAK4 using immunoblotting. The results are shown in FIG. 11. Analysis can see that: the result that the small molecule compound HTS08604 can effectively inhibit the expression of p-ERK1/2 (Thr 202/Tyr 204) and the expression of p-PAK4 is not changed indicates that the HTS08604 can block the ERK/AKT signal pathway and inhibit the phosphorylation level of downstream related proteins in a concentration-dependent manner.
EMT (Epithelial-mesenchymal transition) is an important biological process for acquiring the migration and invasion capacity of malignant tumor cells of Epithelial cell sources, and in order to verify whether the small molecule compound HTS08604 influences the migration and invasion of the cells by regulating the development of the EMT, the inventors collected 0, 1, 5, 10 and 20 mu M HTS08604 to treat esophageal squamous carcinoma cell lines (KYSE 150 and KYSE 450) for 24h and detected the expression levels of N-cadherin, E-cadherin and Vimentin by an immunoblotting method. The results are shown in FIG. 12. Analysis can see that: the expression of E-cadherin is gradually increased and the expression of N-cadherin and Vimentin is gradually reduced along with the increase of the concentration of the compound. This result suggests that HTS08604 influences the migration and invasion of esophageal squamous carcinoma cells by inhibiting EMT progression.
Example 4
Based on example 3, to further explore the targeting of HTS08604 to PAK4, the inventors further conducted related studies by constructing a PAK4 low-expressing cell line, and the specific experimental situation is summarized as follows.
(I) preparing shPAK4 slow virus infection liquid
Firstly, referring to the prior art, a silencing plasmid shPAK4 is prepared (in the construction process, a plurality of different plasmids are designed, and two shPAK4# 5 and shPAK4# 7 plasmids which are self-named are selected for carrying out related experiments).
Subsequently, transfection was performed using the above plasmids, with specific reference to: one day before transfection, in 6cm petri dishes, 1.6X 10 cells were inoculated 6 Culturing HEK293T cells until the cell confluence is 50-60% (for transfection); a transfection solution was prepared in advance (200. Mu.l of the transfection reagent jet buffer, 0.75. Mu.g of PSPA X2, and 0.25. Mu.g of pMD2.G were sequentially added to a 1.5ml EP tube, mixed, centrifuged at 800 rpm for 10 seconds, and then 1. Mu.g of the desired plasmid (Mock, shPAK4# 5, shPAK4# 7) and 4. Mu.l of the desired plasmid were addedl jet primer, mixing uniformly, and standing at room temperature for 10 min); during transfection operation, removing the old culture medium of HEK293T cells, cleaning with 1 × PBS, adding 3 ml of complete DMEM culture medium, adding the prepared transfection solution, mixing uniformly, and culturing in a cell culture box for 4h; after the culture is finished, removing the old culture medium, adding 5ml of complete DMEM culture medium, culturing for 48h in an incubator, collecting virus liquid, and preserving at-80 ℃ for later use; during the experiment, a control group infected with PLKO.1-shmock was designed simultaneously.
Regarding the silencing plasmid involved, the construction process can be roughly referred to as follows:
(1) When the gene silencing primer is constructed for shPAK4# 5, a primer sequence (the primer sequence is designed by the inventor and provided by entrusting Jinzhi company in Shanghai, and the sequence is shown as SEQ ID No. 1-2) is designed as follows:
shPAK4#5-F：5’-CCGGCTGCTGGACGAGTTTGAGAACCTCGAGGTTCTCAAACTCGTCCAGCAGTTTTTG-3’，
shPAK4#5-R：5’-AATTCAAAAACTGCTGGACGAGTTTGAGAACCTCGAGGTTCTCAAACTCGTCCAGCAG-3’；
when the gene silencing primer is constructed aiming at shPAK4# 7, the primer sequence is designed as follows:
shPAK4#7-F：5’-CCGGGACTCGATCCTGCTGACCCATCTCGAGATGGGTCAGCAGGATCGAGTCTTTTTG-3’，
shPAK4#7-R：5’-AATTCAAAAAGACTCGATCCTGCTGACCCATCTCGAGATGGGTCAGCAGGATCGAGTC-3’；
an annealing system is prepared by using the primers, and the 50 mu L annealing system is designed as follows: primer F, 5. Mu.l (20. Mu.M); r primer, 5 μ l (20 μ M); 10 XNEB buffer2, 5. Mu.l; sterile water, 35 μ l; during the annealing operation, the annealing system is put into boiling water and naturally cooled to room temperature (26-27 ℃) to finish the annealing operation.
(2) Carrying out double enzyme digestion on PLKO.1, wherein a 50 mu L double enzyme digestion system is designed as follows: PLKO.1, 3.2. Mu.l (2. Mu.g); 10 XNEB buffer1, 5. Mu.l; 10 × BSA,5 μ l; agel,1 μ l; ecoRI, 1. Mu.l; h 2 O, 34.8. Mu.l; carrying out enzyme digestion for 2h at 37 ℃; after the enzyme digestion is finished, detecting the enzyme digestion product by 0.8 percent agarose gel, and recovering the enzyme digestion product.
(3) And connecting, namely connecting the annealing product with the enzyme digestion product, wherein a 10 mu l connecting system is designed as follows: the annealed product, 2. Mu.l (10. Mu.l of the annealed nucleotide double-stranded product was diluted with 490. Mu.l of sterile water); plasmid PLKO.1 after digestion, 1.8. Mu.l (20 ng); solution I, 5 mu l; sterile water, 1.2 μ l; ligation was carried out at 22 ℃ for 15min.
(4) And (3) transforming and identifying, namely transforming the connecting product in the step (3) into an escherichia coli competent cell by adopting a heat shock method, carrying out resistance screening, amplifying screened and identified correct strains, extracting, recombining and constructing correct plasmids for later use.
Related operations are not described in detail, and conventional operations in the prior art are referred to, and are not described in detail.
(II) infecting esophageal squamous carcinoma cells with lentivirus
In 10 cm culture dish, respectively laying 1.6 × 10 6 Culturing KYSE150 and KYSE450 esophageal squamous carcinoma cells until the cell confluence reaches 60-80%; preparing infection solution (adding 1 ml of the prepared virus solution, 9 ml of complete culture medium and 8 μ l of polybrene in sequence into a 15 ml centrifuge tube, mixing uniformly, and standing at room temperature for 5 min); during infection operation, removing the old culture medium in a culture dish, cleaning by 1 XPBS, adding the prepared and uniformly mixed complete culture medium containing virus solution into the culture dish (simultaneously setting a blank control group without virus), mixing uniformly by light shaking, and culturing in an incubator; after culturing for 24h, removing the complete culture medium containing virus liquid, after 1 XPBS is cleaned, adding a complete 1640 culture medium containing 2 mu g/ml puromycin into a KYSE150 esophageal squamous carcinoma cell culture dish, and adding a complete DMEM culture medium containing 1 mu g/ml puromycin into a KYSE450 cell culture dish; continuously culturing until the cells in the blank control group are completely killed by puromycin; in the culture process, when the cell confluence of esophageal squamous carcinoma cells of the test group (the knockdown group) reaches 80-90%, carrying out passage, and timely collecting the cells to carry out Western blot experiment to detect whether the construction of the PAK4 knockdown cell line is successful.
The detection result of PAK4 protein expression based on Western blot is shown in FIG. 13. As can be seen, compared with Mock, the PAK4 protein is low expressed in the PAK 4-knocked-down esophageal squamous carcinoma cells, which indicates that the PAK 4-knocked-down esophageal squamous carcinoma cell line is successfully constructed.
Referring to the experimental operation, the inventor further evaluates the influence of the small molecule compound HTS08604 on the proliferation of PAK4 knockdown esophageal squamous carcinoma cell lines (KYSE 150 and KYSE 450). The results of the experiment are shown in FIG. 14. Analysis can see that: compared to the control group, 20 μ M HTS08604 had no significant effect on the proliferative capacity of two PAK4 knockdown esophageal squamous carcinoma cell lines (fig. 14A, 14B); the plate cloning experiment result shows that: compared to the control group, 20 μ M HTS08604 had no significant effect on clonogenic capacity of two PAK4 knockdown stable esophageal squamous carcinoma cell lines, while neither the size nor the number of clones were affected by this compound (fig. 14C, fig. 14D); this result further demonstrates that the compound inhibits the proliferation of esophageal squamous cell carcinoma cells by targeting PAK4 protein kinase.
Similarly, referring to the experimental procedures described above, the inventors further evaluated the migration and invasion effects of small molecule compound HTS08604 on PAK4 knockdown esophageal squamous carcinoma cell lines (KYSE 150, KYSE 450). The results of the experiment are shown in FIG. 135. Specifically, the method comprises the following steps: in the control group, 20 μ M of small molecule compound HTS08604 can significantly inhibit the migration and invasion of two esophageal squamous carcinoma cells, while the esophageal squamous carcinoma cells in the shPAK4# 5 and shPAK4# 7 groups have no significant change in migration and invasion after 20 μ M of small molecule compound HTS08604 treatment (fig. 15A, fig. 15B). The result also further indicates that the compound inhibits the migration and invasion capacity of esophageal squamous cell carcinoma cells by targeting PAK4 protein kinase.
Example 5
On the basis of the above experiments, the inventors further performed mouse animal experiments to evaluate the actual control effect of the small molecule compound HTS08604, and the specific experimental procedures and results are summarized as follows.
(I) construction of mouse model for xenotransplantation (CDX)
Mu.l of cell suspension (KYSE 150 cell line, 5X 10 per mouse) was injected per mouse 5 One), injected at the right upper limb scapula of SCID mice; after 1 week of injection, mice were observed and recorded for tumor growth, weekly for miceMonitoring and recording the weight and the tumor volume; when measuring, the length and width of the mouse were measured using a vernier caliper, and the mouse tumor volume = (length × width/2); when the tumor volume reaches 100 mm 3 Then, the tumors were divided into three groups according to their volume: control group (DMSO), 50mg/kg HTS08604, 100 mg/kg HTS08604, 10 animals per group, daily administered by intraperitoneal injection; when the tumor volume of the mice reaches 2000mm 3 In time, mice were sacrificed using cervical dislocation; and taking out the tumor tissue from the mouse under the skin, weighing and photographing, dividing the tumor tissue into two parts after photographing is finished, freezing one part of the tumor tissue at-80 ℃ for later use, and soaking the other part of the tumor tissue in neutral formaldehyde for fixation for immunohistochemical experiments.
(II) construction of in vivo transfer in mice
SCID mice were bred for one week familiar with the environment and evenly divided into three groups based on body weight: control group, 35 mg/kg HTS08604, 70 mg/kg HTS08604, 10 per group; when tumor cells were inoculated, 200. Mu.l of cell suspension (KYSE 150 cell line, 5X 10 cells per mouse) was injected into the middle and rear part of the vein on both sides of the tail of the mouse 5 One); observing the state of the mouse the next day, starting administration when the state is good, and performing intraperitoneal injection administration every day; after the mice are stable for one week, observing the metastasis condition of in vivo tumor cells by using a living animal imager, taking out the mice for photographing at a fixed time every week (1.2 percent of avertin is used for anesthetizing the mice before photographing); when photographing, injecting D-fluorescein potassium salt (10 mu l/g) according to the body weight, injecting the D-fluorescein potassium salt into the abdominal cavity of a mouse for 15min, and then placing the mouse into a living body imaging instrument of the small animal to observe the condition of tumor cells in the body; the transfer was observed for a fixed time until the difference appeared.
Based on the control results in the mouse xenograft (CDX) model, it can be seen that 50mg/kg and 100 mg/kg HTS08604 had no effect on the body weight of the mice (FIG. 16A). HE staining (as is done with reference to conventional procedures) results show that: mice injected with 50mg/kg and 100 mg/kg HTS08604 did not undergo significant pathological changes in their internal organs (fig. 16B). This result indicates that the small molecule compound HTS08604 with the set dose has no toxicity to mice and can be used for subsequent animal experiments. Based on the experimental results of the mouse xenograft (CDX) model (fig. 17), it can be seen that: compared with a control group (DMSO), the intraperitoneal injection of 50mg/kg and 100 mg/kg HTS08604 can obviously inhibit the growth of esophageal squamous carcinoma tumors (figure 17A), and the volume and weight of the tumors are obviously reduced (figure 17B and figure 17C), wherein the inhibition effect of the 100 mg/kg HTS08604 group is more obvious. The inhibition calculations for the two dose groups based on tumor weight show that: 50 The tumor inhibition rates of the HTS08604 group at mg/kg and 100 mg/kg were 56.35% and 80.72%, respectively. This result indicates that HTS08604 can significantly inhibit the growth of esophageal squamous carcinoma in vivo, and the inhibition effect of HTS08604 at 100 mg/kg is better than that at the HTS08604 dose group at 50 mg/kg. The results of the body weight records of the mice of each test group show that (fig. 18): the results, showing that intraperitoneal injection of HTS08604 at this dose was not toxic to mice, showed no significant change in body weight in mice intraperitoneally injected with 50mg/kg and 100 mg/kg HTS08604 compared to the control group.
In the experimental process, in order to verify the change condition of related indexes in regulation of ERK/AKT signal pathways in esophageal squamous cell carcinoma cells by using a small molecule compound HTS08604, the expression conditions of PAK4, p-ERK (Thr 202/Tyr 204) and p-AKT (Ser 473) are further detected by the inventor by using a Western blot technique. The results are shown in FIG. 19. Analysis can see that: compared with a control group (DMSO), 50mg/kg and 100 mg/kg HTS08604 can inhibit an ERK/AKT signal pathway, and mainly shows that the phosphorylation expression levels of ERK and AKT are reduced. However, the expression levels of PAK4 and p-PAK4 in 50mg/kg and 100 mg/kg HTS08604 tumor tissues were not significantly changed, which further suggests that small molecule compound HTS08604 exerts relevant physiological regulatory effects in vivo through PAK4 downstream signaling pathway.
Similarly, the expression of E-cadherin, N-cadherin and Vimentin was examined by Western blot method to evaluate the suppression of EMT progression in esophageal squamous cell carcinoma cells by the small molecule compound HTS08604. The results are shown in FIG. 20. Specifically, the method comprises the following steps: the results of increased expression levels of E-cadherin and decreased expression levels of N-cadherin and Vimentin in the 50mg/kg, 100 mg/kg HTS08604 group compared to the control group (DMSO) indicate that HTS08604 can inhibit the development of EMT in tumor tissues.
The results show that: HTS08604 can block the ERK/AKT signaling pathway and inhibit EMT progression, and to further validate this result, the inventors further examined using IHC method (Immunohistochemistry). The results are shown in FIG. 21. Analysis can see that: HTS08604 can reduce Ki67 expression (Ki 67 is a marker of cell proliferation); at the same time, HTS08604 reduced the phosphorylation expression of ERK, AKT, but the expression of PAK4 and p-PAK4 was unchanged. This result also shows again that: HTS08604 can inhibit the ERK/AKT signaling pathway in CDX tissue; HTS08604 inhibits the expression of N-cadherin and Vimentin, and promotes the expression of E-cadherin. While the development of EMT is mainly manifested by the down-regulation of E-cadherin and the up-regulation of N-cadherin, vimentin, snail, etc. This result suggests that HTS08604 inhibits EMT progression in CDX tissue.
The results of the in vivo transfer model of mice using the small animal living body imager show that (fig. 22): three weeks after cell inoculation, esophageal squamous carcinoma cells mainly metastasize in the lung; compared with a control group (DMSO), the administration group can inhibit the lung metastasis of esophageal squamous cell carcinoma cells, and the high-dose group is further proved to have better inhibition effect by combining the fluorescence expression quantity statistical result and the HE staining result.
Claims (4)
1. The application of the inhibitor for targeted inhibition of PAK4 in preparation of tumor prevention and treatment medicaments is characterized in that the inhibitor for targeted inhibition of PAK4 is a small molecule inhibitor HTS08604 capable of targeted inhibition of PAK 4.
2. Use of an inhibitor of targeted inhibition of PAK4 according to claim 1 for the preparation of a medicament for the prevention or treatment of tumors, in particular esophageal cancer.
3. The use of the inhibitor for the targeted inhibition of PAK4 according to claim 2, wherein the esophageal cancer is esophageal squamous carcinoma.
4. The use of an inhibitor for the targeted inhibition of PAK4 according to claim 1 in the preparation of a medicament for the prevention and treatment of tumors, wherein the small molecule inhibitor HTS08604 is targeted at inhibiting PAK4, and acts to inhibit the proliferation, migration and invasion of cancer cells by blocking the ERK/AKT signaling pathway, inhibiting the EMT progression pattern, and combining the concentration and time-dependent pattern.
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