CN111721932A - Screening method of small molecule compound taking CD133 as target spot and application of small molecule compound in pharmacy - Google Patents
Screening method of small molecule compound taking CD133 as target spot and application of small molecule compound in pharmacy Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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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 and a compound to be tested, using SMM high-throughput screening or affinity experiment detection, and comparing the signal difference between an experimental group and a control group. The invention takes CD133 protein as a target spot and screens target small molecular compounds. The invention also provides the application of the target small molecular compound in preparing anti-tumor drugs, 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, the effectiveness of liver cancer treatment is evaluated, the action mechanism and the law of the compound and protein are clarified on the molecular level, and a foundation is provided for the research and development of new drugs for treating liver cancer.
Description
Technical Field
The invention belongs to the technical field of biochemical pharmacy, and relates to a method for screening anti-liver cancer drugs taking CD133 as a target spot and application thereof.
Background
Liver cancer (HCC) is one of the most common primary liver malignancies, with an increasing trend of morbidity and mortality. At present, the number of liver cancer patients in China is more than half of the number of liver cancer patients in China, and the liver cancer patients become one of the factors seriously threatening the health and life of people. Liver cancer has no typical symptoms in the early stage, and is diagnosed at the later stage of liver cancer, so that the optimal treatment period is missed. The current clinical treatment is also limited, mainly depending on surgical resection, chemotherapy and a few liver transplants. However, the treatment effect is not ideal, and chemotherapy only can kill part of tumor cells, and the residual tumor cells are easy to die again and reburn to form new focuses. Therefore, the deep research on the molecular mechanism of liver cancer development and the search for cost-effective therapeutic drugs are very important for the improvement of clinical diagnosis and treatment schemes and the improvement of patient prognosis.
The stem cell marker CD133 protein, also known as Prominin-1, is a five-transmembrane glycoprotein. Aberrant expression of CD133 was found in different types of tumors. The CD133+ tumor cells have stronger self-renewal capacity, tumorigenic capacity, chemoradiotherapy resistance capacity, migration capacity and the like, play an important role in the relapse and metastasis of tumors, and the characteristics of the tumor cells are the main reasons for difficult tumor healing. In various human liver cancer cell lines, CD133 molecules are used as cell surface markers to sort out CD133+ cell populations and CD 133-cell populations, and the CD133+ cells have stronger tumorigenic capacity through nude mouse subcutaneous tumor formation comparison. The primary cells selected from the liver cancer specimen also express CD133, the expression rate of the CD133 is about 1.3% -13.6%, the cell balls formed by the selected CD133 positive cells in the stem cell culture medium are obviously larger than the negative cells, the subcutaneous tumor forming capability is stronger, and the prognosis of patients with high CD133 expression is worse. In recent years, molecular targeted therapy has been increasingly favored as a new strategy for tumor therapy with therapeutic advantages such as strong specificity and less side effects. Research shows that the CD133 monoclonal antibody can play a therapeutic role by inhibiting the growth of CD133+ liver cancer initiating cells. The fusion toxic protein with CD133 dual specificity targets tumor stem cells, and can obviously inhibit the growth of mouse xenograft lumps. Active small molecule compounds refer to a class of compounds that can cause a phenotypic change in a cell or organism by modulating a biological function of a protein. Many marketed drugs are obtained by modifying the structure of the drugs, so that active small molecule compounds have been a hotspot in research on medicinal chemistry. At present, no clinically effective CD133 inhibitor is found at home and abroad, and due to the lack of a standardized and effective screening method, the screening and the application of the CD133 inhibitor are greatly limited.
Disclosure of Invention
The invention aims to provide a small molecular compound taking CD133 protein as a target spot, a screening method thereof and application thereof in pharmacy.
The invention takes CD133 protein as a target spot, utilizes the small molecule microarray technology to screen out a small molecule compound LDN193189 in high flux, and is verified by in vitro kinase experiments and DARTS experiments; meanwhile, 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, the effectiveness of liver cancer treatment is evaluated, and the action mechanism and the law of the liver cancer and protein are clarified on the molecular level.
First, the present invention provides a method of screening for an anti-tumor compound: and (3) incubating the CD133 with a compound to be detected, detecting by using a small molecule microarray technology or an affinity experiment, and comparing the signal difference between an experimental group and a control group, wherein the target compound is obviously different.
The method specifically comprises the following steps:
preparing a target molecule CD 133;
incubating CD133 with a test compound;
high throughput screening or affinity assay detection using SMM;
and comparing the results of the experimental group and the control group, and screening out the target compound.
In the high-throughput screening step, the compound to be detected is copied on a phenyl isocyanate functional glass slide and a hexyl isocyanate functional glass slide, and the refraction condition of the small molecules combined with the protein can be changed by comparing the light refraction conditions before and after incubation with the C end of the CD133 protein. Preferably, the test compound is exposed to PBS containing BSA to block unprinted isocyanate-functionalized surfaces prior to incubation with CD 133.
In one embodiment of the invention, screening is performed using a DART affinity assay, wherein a test compound is incubated with CD133, followed by addition of a protease, and comparing the properties of CD133 before and after addition of the protease, compounds that interact with CD133 will attenuate the change in CD 133.
In a preferred embodiment of the present invention, a method for SMM high-throughput screening of small molecule microarray targeting human CD133 protein is provided, which is characterized as follows: we picked a pool of 3375 bioactive compounds, including 1053 herbal natural compounds (mostly from herbs), 1527 Food and Drug Administration (FDA) approved drugs, and 795 known inhibitors. Each compound was dissolved in dimethyl sulfoxide at a concentration of 10mM and replicated on phenyl isocyanate functional glass slides and hexyl isocyanate functional glass slides. Exposure to 7600nM BSA in 1 × PBS for 30 min to block unprinted isocyanate functionalized surfaces; small molecules are immobilized on a microarray, on which light is refracted. Then after incubation with the purified CD133 protein C end, the refraction of the small molecule light which is not combined with the protein can not be changed; and the refraction condition of the small molecules after the small molecules are combined with the protein can be changed. Indicating that the compound is possibly a small molecule inhibitor targeting CD133 protein.
The method for screening based on the reaction affinity of the CD133 protein and the small molecule compound is characterized by comprising the following steps: first, the cells were incubated with lysates expressing purified CD133 protein and liver cancer cells Huh7CD133+ for 2 hours at 4 ℃ by LDN193189(1mM, 100. mu.M, 10. mu.M) at different concentrations, and after completion of the incubation, the corresponding enzymes were rapidly added to each tube at a mass ratio of 1:100 (1. mu.g of promoter for 100. mu.g of lysate) and subjected to enzymatic hydrolysis at room temperature (20-25 ℃) for 15 min. Stopping enzymolysis: 0.5M EDTA (pH8.0) was added to the mixture at a mass ratio of 1:10 to stop the enzymatic reaction. If the small molecule compound to be screened is added into cell lysate and then combined with its target protein CD133, the sensitivity to protease is reduced, and the 'protection' effect is strengthened along with the increase of the concentration, which indicates that the two have interaction. Indicating that the compound is possibly a small molecule inhibitor targeting CD133 protein.
Further, the invention provides a small molecule compound with anti-tumor activity, which is called as LDN193189 and has the following structure:
the small molecule compound can be obtained by screening by the method, and can also be artificially synthesized according to a molecular structure.
Correspondingly, the invention provides an anti-tumor kit, and the active ingredient of the anti-tumor kit is LDN 193189.
Further, the invention provides application of the small molecule compound LDN193189 in preparation of antitumor drugs.
In the invention, the antitumor drug targets CD 133.
Wherein, the small molecular compound LDN193189 is an active component of the antitumor drug.
Preferably, the anti-tumor drug is a drug capable of inhibiting tumor cell proliferation and self-renewal.
The anti-tumor drug is a drug capable of inhibiting tumor cell migration.
The anti-tumor drug is a drug capable of inducing tumor cell apoptosis.
The tumor may be any tumor, preferably the tumor is derived from cells positive for CD133 or having an increased expression level.
The invention provides a medicine of CD133 protein targeting hepatoma carcinoma cells, which can be oral liquid, granules, tablets, hard capsules, soft capsules, dropping pills, injections, nano preparations and targeting preparations containing LDN193189 micromolecule compounds.
The development direction of innovative anti-liver cancer drugs is one of the small molecule inhibitors based on CD133 as a target. The invention provides a method for screening an anti-tumor compound, namely incubating CD133 and a compound to be tested, using SMM high-throughput screening or affinity experiment detection, and comparing the signal difference between an experimental group and a control group. The invention takes CD133 protein as a target spot and screens target small molecular compounds. The invention also provides application of the target small molecular compound in preparing antitumor drugs. 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, the effectiveness of liver cancer treatment is evaluated, the action mechanism and the law of the compound and protein are clarified on the molecular level, and a foundation is provided for research and development of new drugs for treating liver cancer.
Drawings
FIG. 1 shows the results of high throughput screening based on small molecule microarray technology; by comparing the image screening results before and after the incubation of the small molecule microarray SMM and the purified CD133 protein, the small molecule LDN193189 combined with the CD133 protein is screened. This compound has a reaction signal in both experiments and the background signal is zero or very weak compared to the reaction signal at the sample spot.
FIG. 2. validation of the interaction between LDN193189 and CD133 protein using DARTS. In FIG. 2A, LDN193189 was incubated with prokaryotic expressed CD133 protein and finally subjected to enzymatic hydrolysis experiments. The concentration of LDN193189 was positively correlated with the signal intensity of the CD133 band. Without LDN193189, CD133 was hydrated by protease. In fig. 2B, LDN193189 was incubated with Huh7CD133+ hepatoma cell lysate and subjected to an enzymatic hydrolysis experiment. When the small molecule LDN193189 is added to the cell lysate, LDN193189 binds to its target protein CD133, thereby decreasing the sensitivity to proteases, and this "protective" effect is enhanced as the concentration of LDN193189 increases.
FIG. 3 shows the growth inhibition effect of small molecular compound LDN193189 on Huh7CD133+ hepatoma cells. FIG. 3A shows the cell culture morphology of small molecule compound LDN193189 before and after treatment. Experimental results show that the morphology of the Huh7 cells is obviously changed after the treatment of the micromolecule LDN193189, the cell density is reduced, the cells are solidified and shrunk, and the volume is reduced. FIG. 3B is a CCK8 proliferation assay with CCK8 assays performed 24h, 48h, 72h after compound treatment. The experimental result shows that LDN193189 has obvious inhibition effect on CD133+ expression cells.
FIG. 4 shows that the small molecular compound LDN193189 inhibits the self-renewal ability of the hepatoma cells of CD133 +. FIG. 4A shows cell balling of Huh7CD133+ hepatoma cells. As shown in the figure, the experimental group LDN193189 cells formed significantly fewer cell balls than the control group, and fig. 4B is a balling experiment of Prf5CD133+ hepatoma cells; the cell balls formed by the LDN193189 cells of the experimental group are obviously smaller in diameter than those of the control group. FIG. 4C shows the soft agar cloning assay of Huh7CD133+, in which the number of stem cell nodules in the same experimental group is significantly less than that in the control group, indicating that LDN193189 inhibits the self-renewal of CD133+ hepatoma cells.
FIG. 5, the small molecular compound LDN193189 inhibits the migration of CD133+ hepatoma cells. Results of Transwell experiments showed that the cells migrating from Huh7CD133+ were significantly reduced after LDN193189 treatment.
FIG. 6. effect of small molecule compound LDN193189 on apoptosis. The control drug Dmso and small molecular compound treated Huh7CD133+ cells after 48h are subjected to annexin V-FITC/PI double staining, and the result shows that LDN193189 can induce Huh7CD133+ hepatoma cells to undergo apoptosis.
FIG. 7 shows that the small molecular compound LDN193189 inhibits the liver cancer. And detecting the expression conditions of p-CD133 and CD133 in the cells and the related proteins of the liver cancer cell cycle, apoptosis and sternness genes after the cells are treated for 48 hours by using a Western blot method. As shown in the figure: after the treatment of a small molecular compound LDN193189, the phosphorylation level of CD133 is obviously reduced, and the expression of an anti-apoptosis gene Bcl-2 protein is increased; the expression level of the cycle-related genes CDK4, CDK6 and P21 gene proteins is reduced, and the expression level of P-src can be inhibited, so that the small molecular compound LDN193189 inhibits the phosphorylation of CD133, further can induce the apoptosis of liver cancer cells, causes cell cycle arrest and inhibits the abnormal activation of P-src.
Detailed Description
The present invention is not limited to the embodiments described above.
The specific conditions and experimental methods not specified in this example are generally performed according to the conventional conditions described in molecular cloning, a laboratory Manual, or according to the conditions provided by the reagent manufacturer.
Example 1 high throughput screening of Small molecule microarrays
Small Molecule Microarrays (SMM) is another high-throughput technology that has rapidly developed over the last decade. SMM refers to a high-density microarray formed by spotting (or printing) and then immobilizing various types of small organic molecules on a solid surface, capable of simultaneously analyzing thousands of biochemical interactions in one step at a time. Microarray of small molecule compounds: the method is mainly used for drug screening and drug discovery. Compound microarrays have the ability to identify and evaluate small molecules, and thus, are 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 from herbal medicine, 1527 drugs approved by the Food and Drug Administration (FDA) and 795 known inhibitors. each compound was dissolved in dmso at a concentration of 10mM and replicated on phenyl isocyanate functionalized slides and hexyl isocyanate functionalized slides.1 x PBS was exposed to 7600nM for 30 minutes to block the unprinted isocyanate functionalized surface, small molecules were immobilized on a microarray and light was refracted thereon. after incubation with the C-terminus of purified CD133 protein, the refraction of light for small molecules that were not bound to protein did not change, while the refraction of small molecules bound to protein did change.
Example 2 screening of Small packets and protein interactions by DARTS technology
DARTS is used as a new drug target identification method, and is a method for verifying the binding of a drug and a target protein mainly by performing proteolysis on the protein which is not bound by the drug. We first incubated the purified CD133 protein and the liver cancer cell Huh7CD133+ lysate with LDN193189(1mM, 100. mu.M, 10. mu.M) at different concentrations for 2 hours at 4 ℃ and after completion of the incubation, the corresponding enzyme was rapidly added to each tube at a mass ratio of 1:100 (1. mu.g of protease for 100. mu.g of lysate) and the enzymatic digestion was carried out for 15min at room temperature (20-25 ℃). Stopping enzymolysis: adding 0.5MEDTA (pH8.0) according to the mass ratio of 1:10 to stop the enzymolysis reaction. When the small molecule LDN193189 is added into a cell lysate, the LDN193189 is combined with a target protein CD133, so that the sensitivity to protease is reduced, and the 'protection' effect is enhanced along with the increase of the concentration of the LDN193189, so that the concentration of the LDN193189 is directly correlated with the signal intensity of a CD133 strip. Whereas, CD133 was hydrolyzed by protease without LDN193189 and no band signal was detected.
Example 3 detection of anti-liver cancer Activity at cellular level
1. Morphological observation of cells
Preparing single cell suspension from CD133+ Huh7 expressing cells according to 3x104And each cell/ml is inoculated into a 6-well plate, 2ml of each well, an administration group and a negative control group are arranged, after 48 hours of culture, the growth state of the cells is observed under an inverted microscope and photographed.
CCK8 proliferation assay
CCK8 cell proliferation detection is carried out by using a 96-well plate, cells expressing Huh7 of CD133+ are digested and suspended in a culture medium containing serum, and the cell density is adjusted to 3x10 by blowing and beating uniformly4And inoculating 3000 cells per well, culturing at 37 ℃, 5% CO2 and saturated humidity, treating with a compound after the cells adhere to the wall, adding 3000 cells into each well of a 96-well plate, and performing CCK8 detection for 24h, 48h and 72h respectively. The result shows that the addition of the small molecular compound LDN193189 can inhibit the proliferation of tumor cells.
3. Stem cell balling experiment
Adherent culture CD133+ expression Huh7 liver cancer was added to the supernatant and the supernatant was added to the digest and centrifuged at 2000rpm for 5min, the supernatant was removed and washed with DMEM/F12 medium (Gibco) containing EGF (Chemicon) 20ng/mL, FGF-2(Chemicon) 20ng/mL, heparin 2 μ g/mL (Sigma) 1:50 vitamin A free B27(Gibco), penicillin 100 μ g/mL and streptomycin 50 μ g/mL, centrifuged at 2000rpm for 5min, the supernatant was removed and washed 2 times with DMEM/F12 medium and the cell number was counted with a cell counter. The cells of the control and experimental groups were aliquoted (100 per well) into low-adsorption 96-well plates (Corning) and suspended in 100. mu.l per well. After being supplemented with 30. mu.l of DMEM/F12 medium every 3 days and cultured for 2 weeks, the number and diameter of formed cell spheres were counted under a microscope, and cell spheres having a diameter of more than 100 μm were used as effective results. The small molecular compound LDN193189 can weaken the self-renewal effect of tumor cells.
4. Soft agar cloning experiments
1) Taking cells in proper growth state, digesting the cells into single cell suspension, counting the single cell suspension by using a cell counter, and blowing the single cell suspension into the single cell suspension by using culture solution for later use.
2) Low melting point agarose solutions were prepared at concentrations of 1.2% and 0.7%, and after autoclaving at high temperatures, the agarose solutions were maintained at 40 ℃ so that they did not coagulate.
3) Mixing 1.2% low melting point agarose solution with 2 × DMEM culture medium (containing 20% fetal calf serum and 2 times of working concentration antibiotics) preheated to 40 deg.C at a ratio of 1:1, spreading on the bottom layer of 6-well plate, cooling and solidifying to obtain the bottom plate.
4) Mixing 0.7% agarose with 2 × DMEM medium (containing 2 times the working concentration of antibiotics and 20% fetal calf serum) at a ratio of 1:1, and adding 3X10 containing (1um compound treated)4And thoroughly and uniformly mixing cell suspension with a cell amount, and adding the cell suspension to the prepared bottom flat plate to obtain the prepared diisetron layer. After the upper agar solidified, the cells were transferred to a cell incubator and cultured for 16 days, during which time observation was made.
5) And (4) acquiring an image by using an imaging system, and calculating the cell clone number and the clone formation rate.
The result shows that the cell clone number and the clone formation rate are reduced by adding the small molecule compound LDN 193189.
5. Apoptosis detection
Preparing single cell suspension from CD133+ Huh7 expressing cells according to 3x104Each/ml was inoculated into 6-well plates (2 ml per well), and the administration group and the negative control group were cultured for 48 hours, digested with trypsin without EDTA, and suspended in a serum-containing medium. Then washed 2 times with PBS (centrifugation at 1000rpm for 2 min.) cells resuspended with 500ul of 1xAnnexin-Bingdingbuffer were added followed by 5ul of Annexin and PI working solution in the chamberIncubation in the dark and warm conditions, and detection by flow cytometry for 15 min.
The result shows that the small molecule compound LDN193189 can promote the tumor cell to generate apoptosis.
Claims (13)
1. A method for screening anti-tumor compounds is characterized in that CD133 and a compound to be tested are incubated, SMM high-throughput screening or affinity experiment detection is used, and the difference of signals of an experimental group and a control group is compared to determine a target compound; the method comprises the following steps:
(1) preparing a target molecule CD 133;
(2) incubating CD133 with a test compound;
(3) high throughput screening or affinity assay detection using SMM;
(4) and comparing the results of the experimental group and the control group, and screening out the target compound.
2. The method of claim 1, wherein in step (3), the compound to be tested is replicated on a phenyl isocyanate functional glass slide and a hexyl isocyanate functional glass slide, and the refraction of the small molecule after being combined with the protein is changed by comparing the refraction of the light before and after incubation with the C-terminal of the CD133 protein.
3. The method of claim 2, wherein the test compound is exposed to PBS containing BSA to block unprinted isocyanate-functionalized surfaces prior to incubation with CD 133.
4. The method of claim 1, wherein the test compound is incubated with CD133 and then the protease is added, and wherein the compound that interacts with CD133 reduces the CD133 change by comparing the CD133 properties before and after the protease is added.
5. The method of claim 1, for use in the preparation or screening of an anti-tumor drug.
6. A small molecule compound with anti-tumor activity, which is characterized in that the structure of the small molecule compound is as follows:
7. the small molecule compound of claim 6, wherein said small molecule compound is screened using the method of claim 1.
8. An antitumor kit, wherein the active ingredient of the kit is the small molecule compound according to claim 7.
9. The use of a small molecule compound of claim 7 in the preparation of an anti-tumor medicament.
10. The use of claim 9, wherein the anti-tumor drug targets CD 133.
11. The use of claim 9, wherein the anti-neoplastic agent is an agent that inhibits tumor cell proliferation, tumor cell migration, tumor cell apoptosis or self-renewal.
12. The use of claim 9, wherein the tumor is derived from a cell that is positive for CD133 or has increased expression.
13. The use of claim 9, wherein the tumor is derived from a CD133 positive hepatoma cell.
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