CN111721932B - Screening method of small molecular compound taking CD133 as target spot and application of small molecular compound in pharmacy - Google Patents

Abstract

The invention belongs to the technical field of biochemical pharmacy, and relates to a method for screening an anti-tumor compound, which comprises the steps of incubating CD133 with a compound to be tested, detecting by using an SMM high-throughput screening or affinity experiment, and comparing the signal difference between an experimental group and a control group. The invention screens target small molecule compounds by taking CD133 protein as a target spot. The invention also provides application of the target small molecular compound in preparing an anti-tumor medicament, and the screened compound is subjected to biological activity detection through a CCK-8 experiment, a stem cell balling experiment and a soft agar cloning experiment, so that the effectiveness of liver cancer treatment is evaluated, the action mechanism and rules of the compound and protein are clarified on a molecular level, and a foundation is provided for research and development of a new medicament for treating liver cancer.

Description

Screening method for small molecule compounds targeting CD133 and its application in pharmaceutical manufacturing

Technical Field

The invention belongs to the technical field of biochemical pharmacy, and relates to a method for screening anti-liver cancer drugs with CD133 as a target and application thereof.

Background Art

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and its incidence and mortality rates are increasing year by year. At present, the number of liver cancer cases in my country accounts for more than half of the country, and it has become one of the factors that seriously threaten people's health and life. There are no typical symptoms in the early stages of liver cancer, and when it is diagnosed, it is basically in the late stage of liver cancer, and the best treatment period has been missed. At present, clinical treatment methods are also relatively limited, mainly relying on surgical resection, chemotherapy and a small number of liver transplants. However, the treatment effect is not so ideal, and chemotherapy can only kill some tumor cells, and the residual tumor cells are prone to resurgence and form new lesions. Therefore, in-depth research on the molecular mechanisms of liver cancer occurrence and development and the search for cost-effective therapeutic drugs are very important for improving clinical diagnosis and treatment plans and improving patient prognosis.

The stem cell marker molecule CD133 protein, also known as Prominin-1, is a five-transmembrane glycoprotein. Abnormal expression of CD133 can be found in different types of tumors. CD133+ tumor cells have stronger self-renewal ability, tumorigenic ability, resistance to radiotherapy and chemotherapy, and migration ability, etc., which play an important role in the recurrence and metastasis of tumors. These characteristics of tumor cells are the main reason why tumors are difficult to cure. In a variety of human liver cancer cell lines, CD133 molecules were used as cell surface markers to sort out CD133+ and CD133- cell populations. Comparison of subcutaneous tumor formation in nude mice showed that CD133+ cells have stronger tumorigenic ability. At the same time, through stem cell sphere experiments, clone formation experiments and proliferation comparisons, it was found that CD133+ tumor cells also have stronger in vitro proliferation and self-renewal abilities. The primary cells sorted from liver cancer specimens also expressed CD133, with an expression rate of about 1.3%-13.6%. The cell spheres formed by these sorted CD133-positive cells in stem cell culture medium were significantly larger than those of negative cells, and the subcutaneous tumor formation ability was also stronger. In addition, patients with high CD133 expression had a worse prognosis. In recent years, molecular targeted therapy has been gradually promoted as a new strategy for tumor treatment, with its strong specificity and few side effects. Studies have shown that CD133 monoclonal antibodies can play a therapeutic role by inhibiting the growth of CD133+ liver cancer initiating cells. The fusion toxin protein with CD133 bispecificity targets tumor stem cells and can significantly inhibit the growth of xenograft tumors in mice. Active small molecule compounds refer to a class of compounds that can cause phenotypic changes in cells or organisms by regulating the biological functions of proteins. Many marketed drugs are obtained by modifying their structures, so active small molecule compounds have always been a hot topic in medicinal chemistry research. At present, no clinically effective CD133 inhibitors have been found at home and abroad. The lack of standardized and effective screening methods has greatly limited the screening and application of CD133 inhibitors.

Summary of the invention

The purpose of the present invention is to provide a small molecule compound with CD133 protein as a target, a screening method thereof and an application of the small molecule compound in pharmaceutical manufacturing.

The present invention uses CD133 protein as a target and utilizes small molecule microarray technology to perform high-throughput screening of the small molecule compound LDN193189, which is verified by in vitro kinase experiments and DARTS experiments. Meanwhile, the bioactivity of the screened compound is tested by CCK-8 experiments, stem cell spheroid experiments and soft agar cloning experiments, the effectiveness of liver cancer treatment is evaluated, and its mechanism and law of action with protein are clarified at the molecular level. The present invention is completed on the above basis.

First, the present invention provides a method for screening anti-tumor compounds: CD133 is incubated with a test compound, small molecule microarray technology or affinity test is used for detection, and the signal difference between the experimental group and the control group is compared. The compound with significant difference is the target compound.

The method specifically comprises the following steps:

preparing target molecule CD133;

Incubate CD133 with the test compound;

Use SMM high-throughput screening or affinity assays;

Compare the results of the experimental group and the control group to screen out the target compounds.

In the high-throughput screening step, the test compound is replicated on a phenylisocyanate functionalized slide and a hexylisocyanate functionalized slide, and the light refraction before and after incubation with the C-terminus of the CD133 protein is compared. The refraction of small molecules will change after binding to the protein. Preferably, the test compound is exposed to PBS containing BSA before incubation with CD133 to block the unprinted isocyanate functionalized surface.

In one embodiment of the present invention, a DART affinity assay is used for screening, wherein the test compound is incubated with CD133, and then a protease is added, and the properties of CD133 before and after the addition of the protease are compared. Compounds that interact with CD133 will attenuate changes in CD133.

In a preferred embodiment of the present invention, a method for high-throughput screening of small molecule microarray SMM targeting human CD133 protein is provided, which is characterized as follows: We selected a library of 3375 bioactive compounds, including 1053 natural compounds of traditional Chinese medicine (mostly from herbs), 1527 drugs approved by the Food and Drug Administration (FDA), and 795 known inhibitors. Each compound was dissolved in dimethyl sulfoxide at a concentration of 10mM and replicated on phenylisocyanate functionalized slides and hexylisocyanate functionalized slides. Expose to 7600nM BSA in 1×PBS for 30 minutes to block the unprinted isocyanate functionalized surface; small molecules are fixed on a microarray and light is refracted on it. After incubation with the purified CD133 protein C-terminus, the refraction of small molecule light that is not bound to the protein will not change; while the refraction of small molecules bound to the protein will change. It shows that the compound may be a small molecule inhibitor targeting CD133 protein.

The method for screening based on the affinity of CD133 protein and small molecule compound reaction is characterized as follows: first, different concentrations of LDN193189 (1mM, 100μM, 10μM) are incubated with the expressed purified CD133 protein and liver cancer cell Huh7CD133+ lysate at 4°C for 2 hours. After the incubation is completed, the corresponding enzyme is quickly added to each tube according to the mass ratio of 1:100 (1μg of pronase forevery 100μg of lysate), and the enzymatic hydrolysis is carried out at room temperature (20-25°C) for 15 minutes. Stop the enzymatic hydrolysis: add 0.5M EDTA (pH8.0) at a mass ratio of 1:10 to stop the enzymatic hydrolysis reaction. If the small molecule compound to be screened is added to the cell lysate and binds to its target protein CD133, thereby reducing the sensitivity to the protease, and as the concentration increases, this "protective" effect is strengthened, indicating that there is an interaction between the two. This indicates that the compound may be a small molecule inhibitor targeting CD133 protein.

Furthermore, the present invention provides a small molecule compound with anti-tumor activity, the small molecule compound is called LDN193189, and its structure is as follows:

The small molecule compound can be obtained by screening using the aforementioned method, or can be artificially synthesized according to the molecular structure.

Accordingly, the present invention provides an anti-tumor kit, the active ingredient of which is LDN193189.

Furthermore, the present invention provides the use of the small molecule compound LDN193189 in the preparation of anti-tumor drugs.

In the present invention, the anti-tumor drug targets CD133.

Among them, the small molecule compound LDN193189 is the active ingredient of anti-tumor drugs.

Preferably, the anti-tumor drug is a drug that can inhibit the proliferation and self-renewal ability of tumor cells.

The anti-tumor drug is a drug that can inhibit the migration of tumor cells.

The anti-tumor drug is a drug that can induce apoptosis of tumor cells.

The tumor may be of any type. Preferably, the tumor originates from cells that are CD133 positive or have increased expression.

The present invention provides a drug targeting CD133 protein of liver cancer cells. The drug can be an oral liquid, granules, tablets, hard capsules, soft capsules, pills, injections, nano preparations and targeted preparations containing LDN193189 small molecule compounds.

Small molecule inhibitors based on CD133 as a target are one of the development directions of innovative anti-liver cancer drugs. The present invention provides a method for screening anti-tumor compounds, that is, incubating CD133 with a test compound, using SMM high-throughput screening or affinity test detection, and comparing the signal difference between the experimental group and the control group. The present invention uses CD133 protein as a target to screen the target small molecule compound. The present invention also provides the use of the target small molecule compound in the preparation of anti-tumor drugs. The biological activity of the screened compounds was tested by CCK-8 experiment, stem cell spheroid experiment and soft agar cloning experiment, the effectiveness of liver cancer treatment was evaluated, and its mechanism and law of action with protein were explained at the molecular level, which provides a basis for the development of new drugs for the treatment of liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. High-throughput screening results based on small molecule microarray technology; by comparing the image screening results before and after incubation of small molecule microarray SMM with purified CD133 protein, the small molecule LDN193189 that binds to CD133 protein was screened. This compound had a reaction signal in both experiments, and the background signal was zero or very weak compared to the sample point of the reaction signal.

Figure 2. DARTS was used to verify the interaction between LDN193189 and CD133 protein. Figure 2A, LDN193189 was incubated with prokaryotic expressed CD133 protein, and finally an enzymatic hydrolysis experiment was performed. The concentration of LDN193189 was positively correlated with the signal intensity of the CD133 band. Without LDN193189, CD133 was all protease-degraded. Figure 2B, LDN193189 was incubated with Huh7CD133+ liver cancer cell lysate and an enzymatic hydrolysis experiment was performed. When the small molecule LDN193189 was added to the cell lysate, LDN193189 bound to its target protein CD133, thereby reducing the sensitivity to proteases, and this "protective" effect was enhanced as the concentration of LDN193189 increased.

Figure 3. The growth inhibitory effect of the small molecule compound LDN193189 on Huh7CD133+ liver cancer cells. Figure 3A shows the cell culture morphology before and after treatment with the small molecule compound LDN193189. The experimental results show that the morphology of Huh7 cells changed significantly after treatment with the small molecule LDN193189, the cell density decreased, the cells shrank, and the volume decreased. Figure 3B is a CCK8 proliferation experiment, and CCK8 detection was performed 24h, 48h, and 72h after compound treatment. The experimental results show that LDN193189 has a significant inhibitory effect on CD133+ expressing cells.

Figure 4. The small molecule compound LDN193189 inhibits the self-renewal ability of CD133+ liver cancer cells. Figure 4A shows the cell spheres of Huh7CD133+ liver cancer cells. As shown in the figure, the cell spheres formed by LDN193189 cells in the experimental group were significantly less than those in the control group. Figure 4B is the sphere formation experiment of Prf5CD133+ liver cancer cells; the diameter of the cell spheres formed by LDN193189 cells in the experimental group was significantly smaller than that in the control group. Figure 4C is a soft agar cloning experiment of Huh7CD133+. In the same experimental group, the number of stem cell spheres was significantly less than that in the control group, indicating that LDN193189 can inhibit the self-renewal of CD133+ liver cancer cells.

Figure 5. The small molecule compound LDN193189 inhibits the migration of CD133+ liver cancer cells. The results of the Transwell experiment showed that after treatment with LDN193189, the number of Huh7CD133+ migrated cells was significantly reduced.

Figure 6. Effect of small molecule compound LDN193189 on cell apoptosis. Huh7CD133+ cells treated with control drug Dmso and small molecule compounds for 48 hours were double stained with AnnexinV-FITC/PI. The results showed that LDN193189 can induce apoptosis of Huh7CD133+ liver cancer cells.

Figure 7. The inhibitory effect of the small molecule compound LDN193189 on liver cancer. Western blot was used to detect the expression of p-CD133, CD133, and liver cancer cell cycle, apoptosis, and stemness gene-related proteins in cells after 48 hours of treatment with small molecule compounds. As shown in the figure: after treatment with the small molecule compound LDN193189, the phosphorylation level of CD133 was significantly reduced, and the expression of the anti-apoptotic gene Bcl-2 protein was increased; the expression levels of cycle-related genes CDK4, CDK6, and P21 gene proteins were downregulated, and the expression level of P-src could be inhibited. Therefore, the small molecule compound LDN193189 inhibits the phosphorylation of CD133, thereby inducing apoptosis of liver cancer cells, causing cell cycle arrest, and inhibiting the abnormal activation of p-src.

DETAILED DESCRIPTION

The specific implementation modes provided by the present invention are described in detail, but the implementation of the present invention is not limited thereto.

Specific conditions and experimental methods not specified in this example are usually implemented according to the conventional conditions described in the Molecular Cloning Experiment Guide, or with reference to the conditions provided by the reagent manufacturer.

Example 1. Small molecule microarray high-throughput screening

Small Molecule Microarray (SMM) is another high-throughput technology that has developed rapidly in the past decade. SMM refers to a high-density microarray formed by spotting (or printing) and then fixing various organic small molecules on a solid surface. It can analyze thousands of biochemical interactions at the same time in one step. Small molecule compound microarray: mainly used for drug screening and drug discovery. Compound microarray has the ability to identify and evaluate small molecules, so it is more useful than other technologies in the pharmaceutical industry.

The present invention prepares a small molecule microarray of 3375 bioactive compounds (, including 1053 natural compounds of traditional Chinese medicine (mostly from herbs), 1527 drugs approved by the Food and Drug Administration (FDA) and 795 known inhibitors. Each compound is dissolved in dimethyl sulfoxide at a concentration of 10mM and replicated on a phenylisocyanate functionalized slide and a hexylisocyanate functionalized slide. Expose to 7600nM BSA in 1×PBS for 30 minutes to block the unprinted isocyanate functionalized surface; the small molecules are fixed on a microarray and refracted when light is shone on it. Then, after incubation with the C-terminus of the purified CD133 protein, the refraction of the light of the small molecules that are not bound to the protein will not change; while the refraction of the small molecules that are bound to the protein will change.

Example 2. DARTS technology to screen small groups and protein interactions

DARTS is a new method for drug target identification. It mainly hydrolyzes proteins that are not bound by drugs by proteases, thereby verifying the binding of drugs to target proteins. We first incubated different concentrations of LDN193189 (1mM, 100μM, 10μM) with the expressed purified CD133 protein and liver cancer cell Huh7CD133+ lysate at 4°C for 2 hours. After the incubation, the corresponding enzyme was quickly added to each tube at a mass ratio of 1:100 (1μg of pronase for every 100μg of lysate), and the enzyme was hydrolyzed at room temperature (20-25°C) for 15 minutes. Stop the enzymatic hydrolysis: Add 0.5MEDTA (pH8.0) at a mass ratio of 1:10 to stop the enzymatic hydrolysis reaction. When the small molecule LDN193189 is added to the cell lysate, LDN193189 binds to its target protein CD133, thereby reducing the sensitivity to proteases, and as the concentration of LDN193189 increases, this "protective" effect is strengthened. It can be seen that the concentration of LDN193189 is positively correlated with the signal intensity of the CD133 band. Without LDN193189, CD133 is hydrolyzed by proteases and no band signal can be detected.

Example 3. Detection of anti-liver cancer activity at the cellular level

1. Cell morphology observation

CD133+ cells expressing Huh7 were made into single cell suspension and inoculated into 6-well plates at 3x10 ⁴ /ml, 2ml per well. A drug-treated group and a negative control group were set up. After culturing for 48 hours, the cells were placed under an inverted microscope to observe their growth status and take pictures.

2.CCK8 proliferation assay

CCK8 cell proliferation assay was performed using a 96-well plate. CD133+ cells expressing Huh7 were digested and suspended in a serum-containing culture medium. The cell density was adjusted to 3x10 ⁴ cells/ml by blowing evenly. 3000 cells were inoculated in each well and cultured at 37°C, 5% CO2 and saturated humidity. After the cells adhered to the wall, the compounds were treated. 3000 cells were added to each well of the 96-well plate and CCK8 assay was performed at 24h, 48h, and 72h. The results showed that the addition of the small molecule compound LDN193189 could inhibit tumor cell proliferation.

3. Stem Cell Sphere Formation Experiment

The supernatant of CD133+ Huh7 liver cancer cells expressing adherent culture was removed and digestion solution was added for 2 min. The cells were centrifuged at 2000 rpm for 5 min and the supernatant was removed. The cells were washed with DMEM/F12 medium (Gibco) containing 20 ng/mL EGF (Chemicon), 20 ng/mL FGF-2 (Chemicon), 2 μg/mL heparin (Sigma), B27 (Gibco) without vitamin A at a ratio of 1:50, 100 μg/ml penicillin and 50 μg/ml streptomycin. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the cells were washed twice with DMEM/F12 medium. The number of cells was counted using a cell counter. The control group and the experimental group were divided into equal amounts of cells (100 per well) into a low-adsorption 96-well plate (Corning), and the cells in each well were suspended in 100 μl. 30 μl DMEM/F12 medium was added every 3 days. After culturing for 2 weeks, photos were taken under a microscope to count the number and diameter of cell spheres formed. Cell spheres with a diameter greater than 100 μm were considered effective results. Small molecule compound LDN193189 can weaken the self-renewal of tumor cells.

4. Soft agar cloning experiment

1) Take cells in an appropriate growth state, digest them into a single-cell suspension, count them using a cell counter, and pipette them into a single-cell suspension with culture medium for later use.

2) Prepare low melting point agarose solutions with concentrations of 1.2% and 0.7%, sterilize them with high temperature and high pressure, and keep them at 40°C so that the agarose solutions will not solidify.

3) Mix 1.2% low melting point agarose solution with 2×DMEM medium (containing 20% fetal bovine serum and 2 times working concentration of antibiotics) preheated to 40°C in a ratio of 1:1, spread on the bottom layer of a 6-well plate, and after cooling and solidification, it becomes the bottom plate.

4) 0.7% agarose and 2×DMEM medium (containing 2 times the working concentration of antibiotics and 20% fetal bovine serum) were mixed in a 1:1 ratio, and then a cell suspension containing 3×10 ⁴ cells treated with (1 μm compound was added thereto, mixed thoroughly, and added to the prepared bottom plate to form a double agar layer. After the upper agar solidified, it was transferred to a cell culture incubator and cultured for 16 days, during which observation was made.

5) Use an imaging system to capture images and calculate the number of cell clones and the clone formation rate.

The results showed that the addition of the small molecule compound LDN193189 reduced both the number of cell clones and the clone formation rate.

5. Apoptosis Detection

CD133+ cells expressing Huh7 were made into single cell suspension, inoculated in 6-well plates at ^3x104 /ml, 2ml per well, and set up drug-treated group and negative control group. After 48h of culture, EDTA-free trypsin was added for digestion and suspended in serum-containing culture medium. Then, it was washed twice with PBS (centrifuged at 1000rpm for 2min). 500ul of 1xAnnexin-Bingdingbuffer was added to resuspend the cells, and then 5ul of Annexin and PI working solution were added, incubated at room temperature in the dark, and detected by flow cytometry for 15min.

The results showed that the small molecule compound LDN193189 could promote apoptosis of tumor cells.

Claims (2)

1. Of the following structureApplication of small molecular compound in preparing tumor medicine for resisting CD133 positive liver cancer;

the antitumor drug targets CD133:

。

2. The use according to claim 1, wherein said small molecule compound is obtained by screening by the following method: incubating CD133 with the test compound, detecting by using SMM high throughput screening or affinity assay, comparing the signal difference between the experimental group and the control group, and determining the target compound;

The method comprises the steps of:

(1) Preparing a target molecule CD133;

(2) Incubating CD133 with a test compound;

(3) Detection using SMM high throughput screening or affinity experiments;

(4) Comparing the results of the experimental group and the control group, and screening out a target compound;

The compound to be tested in the step (3) is replicated on a phenyl isocyanate functional glass slide and a hexyl isocyanate functional glass slide, and the light refraction conditions before and after incubation with the C end of the CD133 protein are compared, so that the refraction conditions of small molecules after combination with the protein are changed; prior to incubation of test compounds with CD133, exposure to BSA-containing PBS blocked the unprinted isocyanate-functionalized surface; the test compound is incubated with CD133, then protease is added, and the CD133 change is attenuated by the compound interacting with CD133, as compared to the nature of CD133 before and after protease addition.