Application of tryptophan derivative probe of parthenolide in inhibiting action target of glioma cell proliferation

Abstract

The invention relates to an application of a parthenolide tryptophan derivative probe in inhibiting an action target of glioma cell proliferation, in particular to an application of a parthenolide tryptophan derivative probe PDTry-TZ in determining a parthenolide tryptophan derivative as the action target of inhibiting glioma cell proliferation. According to the present invention, the probe of the parthenolide tryptophan derivative is synthesized, the probe is used for determining the action target of the parthenolide tryptophan derivative, and the possible action target of the probe is found through a series of experiments;

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to an application of a tryptophan derivative probe of parthenolide in determining that the tryptophan derivative of parthenolide serves as a target for inhibiting the proliferation of glioma cells. Background Art

[0002] As we all know, targets are the source and key biological basis for the therapeutic effects of drugs. Therefore, the identification and functional research of drug targets are of great guiding significance for clarifying the mechanism of action of drugs. At the same time, the discovery of new drug targets is also the cornerstone of innovation in the pharmaceutical industry and the focus of competition among major international pharmaceutical companies.

[0003] Active ingredients of traditional Chinese medicine refer to monomeric compounds with medical efficacy or physiological activity in traditional Chinese medicine. Traditional Chinese medicine has a wide range of sources, complex ingredients, and a wide variety of active ingredients. It is an important way for humans to obtain active ingredients, lead compounds, and prepare drugs.

[0004] The chemical proteomics platform can discover the protein targets of natural products of traditional Chinese medicine, transform natural products of traditional Chinese medicine into multifunctional chemical probes with similar activities, directly capture active molecule binding proteins in living cells or diseased tissues, separate and enrich target proteins with the help of bioorthogonal coupling reactions, and identify them using high-resolution mass spectrometry with the accuracy of binding pockets, providing more comprehensive and accurate information for revealing the mechanism of action of active ingredients of traditional Chinese medicine.

[0005] Since the American Association for Cancer Research (AACR) launched a large-scale anti-tumor drug screening program in 1960, 67% of the anti-cancer drugs are natural products or derivatives of natural products.

[0006] Terpenoids are a class of natural products that are most widely distributed in nature and have diverse structural types. Common plant terpenoid components include terpenes, steroids, saponins, cardiac glycosides and carotenoids.

[0007] Parthenolide is a germarane-type sesquiterpene lactone compound derived from medicinal plants. Parthenolide has certain anti-tumor activity. However, its target of action is still unclear. Summary of the invention

[0008] The present inventors have discovered that parthenolide tryptophan derivatives can be used to inhibit the growth of tumor cells.

[0009] The present invention provides an application of a compound in determining a parthenolide tryptophan derivative as a target for inhibiting the proliferation of glioma cells.

[0010] The glioma cell lines are: U87 human brain glioma cells, U251 human glioma cells, and A172 human glioblastoma cells.

[0011] Experiments have shown that parthenolide has pharmacological activity against a variety of cancer cells, showing broad-spectrum anti-cancer characteristics, especially in the treatment of various solid tumors and blood diseases. It also has anti-inflammatory, redox regulation and epigenetic activity, as well as cytotoxicity to cancer stem cells, but is harmless to normal stem cells. This has made parthenolide PTL the focus of medical researchers, indicating that it has great potential to become a first-line anti-cancer drug. However, the water solubility of parthenolide compounds is poor. The present invention increases its water solubility and anti-tumor activity by introducing amino acids, among which the tryptophan derivative PDTry (IC 50 =2.381µM / ml), which has a very good inhibitory effect on the growth of glioma cells, but its target is still unclear. By modifying the drug and introducing a small molecule probe, the tryptophan derivative probe PDTry-TZ (IC 50 =3.352µM / ml), using proteomics to study its molecular mechanism of action is currently a way to confirm its target. Based on this, the present invention uses the tryptophan derivative of parthenolide PDTry as a reactant to synthesize a probe of the tryptophan derivative of parthenolide, and uses proteomics to study it as a tool for "hooking" the target, and successfully finds its possible target. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The silver staining result of the protein sample provided in the embodiment of the present invention; Figure 2 The figure shows the rhodamine test (left picture) and Coomassie brilliant blue staining (right picture) provided in the embodiments of the present invention; Figure 3 It is a volcano map of enriched proteins provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0013] The invention is illustrated by the following examples.

[0014] Target research of parthenolide tryptophan derivatives based on chemical proteomics: Experimental research objects, reagents and instruments: Human glioblastoma astrocytoma cells U87MG were cryopreserved by the School of Pharmacy and Pharmaceutical Sciences, Shandong First Medical University.

[0015] Preparation of stock solution: Accurately weigh 2 mg of the tryptophan derivative probe PDTry-TZ probe molecule of parthenolide, add DMSO solution to dissolve it, and prepare a 40 mM stock solution. The stock solution is divided and frozen in a -80℃ refrigerator. Before the experiment, it is diluted with culture medium to the required concentration according to the experimental design.

[0016] Table 1 Main reagents Table 2 Main instruments and equipment for the experiment Evaluation of the cytotoxic effect of the tryptophan derivative probe PDTry-TZ probe molecule on parthenolide: Take U87 cells in the logarithmic growth phase, count the cells after digestion, and dilute the cell suspension to a concentration of 1×10 5 / ml, inoculated into 96-well plates, with a volume of 100µL added to each well, and the medium was replaced after 12h. 80µL of fresh complete medium was added to the wells, and the pre-prepared drug storage solution was diluted according to the required concentration, diluted to 200, 100, 50, and 25µmol / L, respectively, and 20µL was added to each well, so that the final concentration was 40, 20, 10, and 5µmol / L per well. Five replicate wells were set for each concentration, and a control group containing only cells and DMSO without drugs was set up; a complete culture medium without cells was set up as a blank group. After culturing in a 37°C, 5% CO2 incubator for 24h, 10µL of CCK-8 solution was added to each well, incubated in the incubator for 2h, and the absorbance (OD) value at 450nm was measured with an enzyme reader, and the survival rate of various cell lines was calculated according to the following formula, and the corresponding IC 50 The experiment was repeated 3 times.

[0017] Cell viability = [(OD of experimental well - OD of blank well) / (OD of control well - OD of blank well)] × 100%.

[0018] In situ labeling of cancer cell lines using molecular probes - cell culture Take the normally cultured U87MG cells in the logarithmic growth phase, replace the culture medium containing special markers with their culture medium (light or heavy), and subculture when the cell confluence reaches 80%-90%. Use the special labeled complete medium to culture and subculture the cells until the 6th generation of cell confluence reaches 80-90%, collect the cells and store them at -80℃. At this time, the proteins in the cells are basically labeled with the corresponding isotopes, and the labeling efficiency reaches 95%. Three plates of light and heavy labeled cells are required.

[0019] Detection of cell labeling efficiency: After culturing U87MG cells to the 6th to 7th generation, samples were sent for testing of labeling efficiency.

[0020] Take 300ul of the labeled cells respectively, centrifuge and discard the supernatant, wash with PBS into 1.5ml EP tubes, centrifuge at 3000rpm for 2min, repeat the wash twice, add 150ul cell lysis buffer NP40, lyse on ice for 0.5 hour, vortex and oscillate every 10 minutes, centrifuge at 12000rpm at 4℃ for 10 min, retain the supernatant, and send the sample after BCA protein determination.

[0021] In-situ target fishing: 1) Set up a binding group (probe 10mM), a blank group, and a competition group (probe 10mM + 20mM small molecule compound).

[0022] 2) Inoculate U87MG cells onto a 10cm dish. When the cell confluence reaches 80%-90%, add small molecules and incubate for 2 hours. Set up the following groups: blank group (only given the same volume of DMSO), combination group, and competition group. First, administer the probe, and then add 2 times the volume of PDTry 30 minutes later. When administering to cells, to prevent insolubility, mix with the culture medium in advance and then add after beating evenly.

[0023] 3) Irradiate with UV light for 10 minutes (UV light wavelength is 365nm). After discarding the culture medium, add 1ml PBS to wash once and discard it, then add 2ml PBS to irradiate with UV light and open it in a closed space (Note: place the dish on ice).

[0024] 4) Collect cells, scrape them directly with a scraper, place them in a 15ml centrifuge tube, centrifuge at 2500rpm for 2min, discard the supernatant, add 1ml PBS to blow down the cells and transfer them to a 1.5ml EP tube and centrifuge at 3000rpm for 2min, repeat the wash twice. The cells can be removed from the PBS and frozen at -80℃ for later use.

[0025] 5) After removing PBS, add 500ul of NP40 lysis buffer, vortex, place the resulting suspension on a shaker in an ice bath for 30min, repeat the vortex twice, centrifuge the suspension at 12000rpm at 4℃ for 10-15min, and retain the supernatant. (The lysis time can be longer to ensure lysis efficiency).

[0026] 6) Determine the protein content and adjust the protein to the same concentration. Each sample should preferably be greater than 0.7 mg.

[0027] The BCA concentration gradient was set to 0, 1, 2, 4, 8, 12, 16, 20, and the corresponding PBS concentration gradient was 20, 19, 18, 16, 12, 8, 4, 0. Solution A and solution B in the BCA protein concentration assay kit were mixed at a ratio of 50:1, and a 96-well plate was used to configure the assay standard curve.

[0028] Sample operation: Take 18µL of PBS buffer and add 2ul of sample to each 96-well plate, i.e. dilute 10 times. Each well still needs 200µL of BCA working solution, which is divided into blank group, binding group and competition group. 2.5ml of A solution and 50ul of B solution are required. Incubate at 37℃ for 30min and measure the OD value. After making the standard curve, adjust the protein. Ensure that the protein of each sample is greater than 0.7mg. The operation can be done in EP tubes in advance, and the samples must be parallel.

[0029] 7) Click reaction (1) Add 2.9 µL Biotin-PEG3-N3 (20 mM in DMSO) to each tube and vortex; (2) Add 11.6 µL TCEP to each tube without vortexing (TCEP is prepared immediately: 14.4 mg / mL, prepared with water); (3) Add 17.5 µL (3.4 mM) Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine to each tube and vortex; (Prepared, add 1.75 µL).

[0030] (4) Add 11.6 µL copper(Ⅱ) sulfate (50 mM in water, 12.5 mg / ml) to each tube and vortex. (5) Leave at room temperature for 1-2 hours (vortex after 30 minutes) until the protein begins to precipitate; (6) Centrifuge for 10 min at 6500 g at 4°C to form a protein pellet. Remove the supernatant, add 500 μl of ice-cold methanol to each tube, sonicate for 3-4 seconds, and centrifuge at 6500 g at 4°C for 4 min. (7) Remove the supernatant and wash repeatedly with 500 µL of ice-cold methanol; (8) Remove the supernatant and add 0.2 ml 1.2% SDS / PBS; (9) Add 1 ml of PBS and dilute to 0.2% SDS; Samples can be stored at -80°C for several days.

[0031] 8) Affinity purification: Add 15ul streptavidin beads to the sample, wash with PBS for 3 times; then add the treated protein sample and incubate at 4℃ overnight; at this time, please note: if it is magnetic beads, wash with SDS / PBS on the magnetic stand without centrifugation. Centrifuge at 1400rpm for 3min and remove the supernatant; wash with 0.2% SDS / PBS, rotate for 10min, centrifuge at 1400g for 3min, remove the supernatant; use PBS and rotate for 1min, centrifuge at 1400rpm for 3min, remove the supernatant, and repeat this process 3 times. Perform silver staining analysis.

[0032] Rhodamine probe validation (1) Add 2.9 µL of rhodamine (20 mM, in DMSO) to each tube and vortex; (2) Add 11.6 µL TCEP to each tube without vortexing (TCEP is prepared immediately: 14.4 mg / mL, prepared with water); (3) Add 17.5 µL of 3.4 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine to each tube and vortex; (the actual concentration is 1.75 µL) (4) Add 11.6 µL copper(Ⅱ) sulfate (50 mM in water, 12.5 mg / ml) to each tube and vortex. (5) Leave at room temperature for 1-2 hours (vortex after 30 minutes) until the protein begins to precipitate; (6) Centrifuge for 4 min at 6500 rpm at 4°C to form a protein pellet. Carefully remove the supernatant, add 500 µL of ice-cold methanol to each tube, sonicate for 3-4 s or vortex or blow directly to break up the protein pellet, and centrifuge again at 4°C and 6500 rpm for 4 min. (7) Remove the supernatant and wash repeatedly with 500 µL of ice-cold methanol; (8) Remove the supernatant, add PBS according to the amount of sample, dilute to 1X with 6X loading buffer, and boil the sample in a metal bath at 98°C for 10 min. Samples can be stored at -80°C for several days.

[0033] Add 80µL PBS to each sample. If there are 3 samples, mix 240ul PBS with 48µL of loading buffer diluted five times, add to 3 samples respectively, and boil the samples. Boil the samples in a metal bath at 98℃ for 10min. Protein enrichment results of parthenolide tryptophan derivative probe: like Figure 1 As shown in the figure, before high-resolution mass spectrometry proteomics, SDS-polyacrylamide gel electrophoresis and silver staining were performed to prepare the protein sample. These two steps were used to confirm the authenticity of the soluble protein sample.

[0034] The silver staining results of the above protein samples are shown from left to right: Marker, blank group, binding group (PTL-D-probe 10mM), competition group 1 (PTL-D-probe 10mM+20mM PTL-D-tryptophan derivative), competition group 2 (PTL-D-probe 10mM+10mM PTL-D-tryptophan derivative). Specific protein bands can be observed, proving that there is no problem with this batch of protein samples.

[0035] Secondly, if Figure 2 As indicated, co-incubation with rhodamine ( Figure 2 Left figure), from the pull-down results, the binding group (PTL-D-probe 10mM) showed obvious fluorescent bands, and the PTL-D-tryptophan derivatives in the competition group competed to bind to some proteins, but because the probe was not connected, the fluorescence intensity should be significantly weakened, consistent with the rightmost band; the blank group had no obvious fluorescence intensity. Prove that this probe is effective. Next, the enriched protein was digested with trypsin and digested into peptides to prepare the peptide samples required for high-resolution mass spectrometry, and then tested.

[0036] Proteomic analysis based on high-resolution mass spectrometry detection: like Figure 3 As shown, the present invention cuts the gel near 100kDa, 75kDa, 60kDa, and 45kDa in the pull-down results, prepares polypeptide samples for high-resolution detection, and performs database search to filter out possible contamination and false positive proteins. The number of remaining proteins is 1351, and this volcano map is obtained. The inventor selected possible potential protein targets (red parts) with obvious differences. Seven protein targets were selected from the upregulated, downregulated, and overlapping proteins, and these potential target proteins need to be further verified later.

[0037] The present invention synthesized a probe of tryptophan derivatives of feverfew and applied it to determine the action targets of tryptophan derivatives of parthenolide. Through a series of experiments, the inventors found that the possible action targets are shown in Table 3 below: Table 3 Proteomics enriched protein screening list

Claims

1. Use of a tryptophan derivative probe PDTry-TZ of parthenolide of the following formula (1) in determining the tryptophan derivative of parthenolide of the following formula (2) as a target for inhibiting the proliferation of glioma cells; 。 2. The use according to claim 1, characterized in that: The glioma cell lines are: U87 human brain glioma cells, U251 human glioma cells, and A172 human glioblastoma cells.

Images