A GRB2 small molecule inhibitor and its preparation method and application

Abstract

The present invention discloses a GRB2 small molecule inhibitor and a preparation method and application thereof. The phenolic hydroxyl group of hydroxycamptothecin is structurally modified, and 3-(2-bromoethyl)-3-(butane-3-yn-1-yl)-3H-diaziridine is reacted with hydroxycamptothecin to obtain the GRB2 small molecule inhibitor, and the new compound is named HCPT-pt. Experimental verification shows that the compound HCPT-pt is a GRB2 small molecule inhibitor, and its ability to kill esophageal cancer cells is stronger than that of hydroxycamptothecin, and it can be used as a drug for the treatment of a wide range of tumor diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a GRB2 small molecule inhibitor and a preparation method and application thereof. Background Art

[0002] Endoscopic resection is the main treatment for early esophageal cancer, while surgery and chemoradiotherapy are the main treatments for advanced esophageal cancer. However, patients with advanced esophageal cancer usually have inherent resistance to systemic therapy, resulting in a poor prognosis.

[0003] In recent years, some targeted drugs such as PD-L1 inhibitor pembrolizumab have been developed and approved for the treatment of esophageal cancer and have achieved good results, but complete recovery cannot be achieved due to drug resistance. Therefore, there is an urgent need to develop novel and effective targeted therapeutic drugs to improve the anti-cancer efficacy of esophageal cancer.

[0004] The GRB2 (Growth factor receptor-bound 2) protein consists of 217 amino acids and has a molecular weight of approximately 25 kDa. GRB2 was originally discovered to be a missing link between the epidermal growth factor receptor (EGFR) and the Ras mitogen-activated protein kinase (MAPK) pathway. The most significant feature of the GRB2 protein structure is that it contains a central SH2 domain and two SH3 domains on both sides. The SH3 domain contains approximately 50 amino acids and can recognize proline-rich proteins such as SOS (Son of Sevenless), which plays an important role in regulating the function of GRB2 and the signaling pathways in which it is located. In addition, SOS is a guanine nucleotide release factor that converts membrane-bound Ras from an inactive GDP-bound form to an active GTP-bound form.

[0005] It has been reported (Zu Ye, et. al., GRB2 enforces homology-directed repairinitiation by MRE11, Sci Adv. 2021 Aug 4;7(32):eabe9254. doi: 10.1126 / sciadv.abe9254. Print 2021 Aug.) that GRB2 is highly expressed in a variety of tumors and promotes tumor invasion, growth, metastasis, and drug resistance. For example, the same situation is found in esophageal cancer, gastric cancer, diffuse large B-cell lymphoma, pancreatic cancer, skin melanoma, testicular cancer, etc. In addition, the activation of GRB2 is negatively correlated with the prognosis of tumor patients. These findings suggest that GRB2 is a potential target for tumor treatment.

[0006] Hydroxycamptothecin is an alkaloid extracted from the seeds or root bark of the deciduous plant Camptotheca acuminata of the Davidiaceae family. Its CAS number is 19685-09-7, and its molecular structure is shown in Formula I.

[0007] Formula I,

[0008] Currently, hydroxycamptothecin is used to treat gastric cancer, liver cancer, head and neck cancer, and leukemia.

[0009] For example, the invention application with publication number CN1654039A discloses an oral preparation of hydroxycamptothecin and a preparation method thereof. Hydroxycamptothecin is used to treat sarcoma, lung cancer, etc.

[0010] The invention application with publication number CN102988366A discloses the use of hydroxycamptothecin and a nasal administration agent for treating intracranial tumors.

[0011] It is of positive significance to develop more active anticancer drugs based on hydroxycamptothecin. Summary of the invention

[0012] In view of the above-mentioned deficiencies in the prior art, the present invention provides a GRB2 small molecule inhibitor and a preparation method and application thereof.

[0013] The present invention first provides a small molecule inhibitor of GRB2, named HCPT-pt, with a structural formula as shown in Formula II,

[0014] Formula II.

[0015] The present invention further provides the use of the GRB2 small molecule inhibitor in the preparation of an anti-tumor drug. Preferably, the tumor type is a GRB2-positive tumor. More preferably, the tumor type is esophageal cancer, gastric cancer, diffuse large B-cell lymphoma, pancreatic cancer, skin melanoma or testicular cancer.

[0016] The present invention also provides an anti-tumor drug, wherein the active ingredient is the GRB2 small molecule inhibitor. Preferably, the tumor type is a GRB2-positive tumor. More preferably, the tumor type is esophageal cancer or gastric cancer.

[0017] The present invention also provides a method for preparing the GRB2 small molecule inhibitor, wherein the phenolic hydroxyl group of hydroxycamptothecin is structurally modified, and 3-(2-bromoethyl)-3-(but-3-yn-1-yl)-3H-diaziridine is reacted with hydroxycamptothecin to obtain the GRB2 small molecule inhibitor.

[0018] The present invention modifies the structure of the phenolic hydroxyl group of hydroxycamptothecin, reacts with halogenated hydrocarbons to form an ether, and then connects a small molecule to obtain a new compound HCPT-pt. Experimental verification shows that the compound HCPT-pt is a new GRB2 small molecule inhibitor, and has a stronger ability to kill esophageal cancer cells than hydroxycamptothecin, and can be used as a drug for a wide range of tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the reaction formula of the compound HCPT-pt of the present application.

[0020] Figure 2 This is the hydrogen spectrum verification result of the new compound HCPT-pt.

[0021] Figure 3 This is a graph showing the half-maximal inhibition concentration test results of hydroxycamptothecin and HCPT-pt in Example 2.

[0022] Figure 4 This is a flow chart for target protein identification in Example 3.

[0023] Figure 5 This is a scatter plot with fold change in Example 3. GRB2 protein is highly enriched.

[0024] Figure 6 To detect the binding ability of HCPT-pt and GRB2 protein. Figure 6 A in the figure is the detection result of the protein expression level of GRB2 in cells after HCPT-pt treatment with temperature; Figure 6 B is the grayscale value of protein expression quantified by ImageJ, and three independent samples were repeated for each temperature.

[0025] Figure 7 Constructed assay results graph for the GRB2 knockout KYSE150 cell line.

[0026] Figure 8 Figure 2. Colony formation assay of KYSE150 wild-type and GRB2 knockout cells treated with the indicated concentrations of HCPT-pt. Figure 8 A in the figure is a representative picture of clone formation experiment; Figure 8 B in the figure is the number of clones quantified by ImageJ. ns means not significant, *P<0.05, ***P<0.001, ****P<0.0001, the same below.

[0027] Fig. 9 The scratch assay was used to detect the migration ability of KYSE150 wild-type and GRB2 knockout cells after being treated with different concentrations of HCPT-pt for 48 hours. Fig. 9 A in the figure is a representative picture of the scratch experiment; Fig. 9 B in the figure is a quantitative analysis of the healing rate of the scratch test.

[0028] Fig.10 KYSE150 wild-type cells and GRB2 knockout cells were treated with different concentrations of HCPT-pt for 48 hours. Fig.10 A in the figure is the Transwell assay to analyze the invasion ability of cells after HCPT treatment; Fig.10 B in the figure shows the number of cells that penetrated the chamber quantified using ImageJ. DETAILED DESCRIPTION

[0029] Example 1

[0030] The molecular structure of hydroxycamptothecin is shown in Formula I.

[0031] Formula I,

[0032] The phenolic hydroxyl group of hydroxycamptothecin is structurally modified and reacted with halogenated hydrocarbons to form an ether, thereby connecting a small molecule. Figure 1 shown.

[0033] Hydroxycamptothecin (1 eq, Figure 1 Compound A) and 3-(2-bromoethyl)-3-(but-3-yn-1-yl)-3H-bis(aziridine) (Macklin, Cat. No. 2432830-88-9, Figure 1 Compound B (2 eq) was dissolved in DMSO and K was added dropwise to the stirred solution. 2 CO 3 Solution (1~1.5eq). The reaction mixture was stirred at room temperature for 24 hours and then diluted with 10% (volume concentration) MeOH in dichloromethane (80mL). The resulting suspension was filtered and the residue was washed with 10% (v / v) MeOH in dichloromethane. The solid material was washed with saturated K 2 CO 3 , washed with saturated brine, and 2 SO 4The product was dried on a plate, the solvent was evaporated and removed, hexane: ethyl acetate (V / V 1:1) was added to the product for re-suspending, the suspension was filtered, and washed twice with hexane: ethyl acetate (V / V 1:1), filtered and dried to obtain a pure light yellow solid product. The synthesized new small molecule compound is abbreviated as "HCPT-pt" and has a structural formula as shown in Formula II. Proton spectrum data: 1H NMR (400 MHz, Methanol-d4) δ8.45 – 8.31 (m, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 7.42 (dd, J =9.3, 2.8 Hz, 1H), 7.25 (d, J = 2.7 Hz, 1H), 5.39 (dd, J = 96.5, 16.2 Hz, 2H),5.20 (s, 2H), 3.95 (t, J = 6.1 Hz, 2H), 2.14 (t, J = 2.7 Hz, 1H), 2.02 (td, J= 7.4, 2.7 Hz, 2H), 1.92 (t, J = 6.0 Hz, 2H), 1.87 (dd, J = 7.4, 3.9 Hz, 2H), 1.66 (t, J = 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H), as Figure 2 shown.

[0034] Formula II.

[0035] Example 2

[0036] A concentration gradient containing a concentration gradient range of 0.0625-12.8 μM HCPT / HCPT-pt was set up using a geometric progression (logarithmic value 2). Wild-type esophageal cancer cell KYSE150 was seeded in a 96-well plate at a density of 2000 cells / well. The survival rate of 2000 cells per well was observed after 48 hours. The cell viability was detected by CCK8 method. The absorbance of each well was detected at a wavelength of 490 nm and the relative cell viability was calculated (relative cell viability %=100%×(OD 待测样本 -OD 空白本底 ) / (OD 对照样本 -OD 空白本底 ), three independent samples were repeated for each concentration.

[0037] The results are as follows Figure 3As shown, the half inhibition concentration of HCPT-pt was found to be 207.6nM, and the half inhibition concentration of HCPT-pt was found to be 124.3nM. At the same concentration, HCPT-pt has a stronger ability to kill esophageal cancer cells than HCPT-pt. We determined that the half inhibition concentration of HCPT-pt is about 200nM, and this concentration is used as the standard for subsequent experiments.

[0038] Example 3

[0039] First, 200 nM HCPT-pt was mixed with 1×10 7 A number of esophageal cancer cells (KYSE150) were co-incubated for 2 hours, and then irradiated at 365nm UV spectrum for 10 minutes for UV cross-linking to combine the potential target protein with HCPT-pt. Then, the cells were ultrasonically lysed with an energy parameter of 10%, and the supernatant was centrifuged to obtain a soluble protein solution. The group without photocross-linking reaction was set as the control group.

[0040] Prepare click reaction reagents: biotin (0.25 μL, 10 mM); CuSO 4 (0.5μL, 50mM); TBTA (1.5μL, 1.7mM, CAS No. 510758-28-8); TCEP (DTT, 0.5μL, 50mM).

[0041] Freshly pre-mixed click reaction reagents were added to the two protein solutions to connect the biotin tags, and the solutions were incubated at room temperature in the dark for 1 hour.

[0042] Then, Strep magnetic beads were added for co-incubation for 1 hour, and then the beads were washed three times with PBS and sent for mass spectrometry. Compared with the control group, the proteins with increased protein abundance in the experimental group are potential drug targets. Figure 4 shown.

[0043] After mass spectrometry analysis, we found that GRB2 protein was highly enriched as a highly reliable protein. Therefore, we speculated that GRB2 is a potential target of HCPT-pt (e.g. Figure 5 as shown).

[0044] Example 4

[0045] The binding between HCPT-pt and GRB2 in cells was analyzed by cellular thermal shift assay (CETSA).

[0046] Each group will have 2×10 7Esophageal cancer cells (KYSE150) were seeded in 10 cm culture dishes. After 24 h of culture, cells were pretreated with 10 μM MG132 for 1 h, washed with PBS, and collected by trypsin digestion. The samples were centrifuged at 12,000 rpm for 5 min at room temperature, gently resuspended with 1 mL PBS, and centrifuged again to discard the supernatant. The cell pellet was resuspended with 1 ml of RIPA lysis buffer (purchased from Applygen), and 10 μL of protease inhibitors (purchased from Dawen Biotechnology) were added and lysed on ice for 10 min. The samples were then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was transferred to a new PCR tube. For the experimental sample group, HCPT-pt (small molecule drug dissolved in DMSO) was added to a final concentration of 200 nM. For the control sample group, the same volume of carrier solvent (DMSO) was added. Each group consisted of one control aliquot and one experimental aliquot, which were heated for 3 min at 40°C, 43°C, 46°C, 49°C, 52°C, 55°C. Finally, the samples were subjected to Western blot analysis.

[0047] The results are as follows Figure 6 As shown, Figure 6 A in the figure is the detection result of the protein expression level of GRB2 in cells after HCPT-pt treatment with temperature; Figure 6 B in the figure is the grayscale value of protein expression quantified by ImageJ. Three independent samples were repeated at each temperature. The results showed that the thermal stability of GRB2 protein treated with HCPT-pt was significantly higher than that of the control group as the temperature increased. This further confirmed that GRB2 is the target of HCPT-pt.

[0048] Example 5

[0049] Construct a GRB2 knockout esophageal cancer cell line (KYSE150). First, design the knockout plasmid sgRNA fragment based on the CRISPR-Cas9 system, and the corresponding target sequence is as follows:

[0050] 5′-AGATGGAGCCGGGAAGTACT-3′.

[0051] The target fragment sgRNA was constructed into the CRISPR-Cas9 system to construct a CRISPR-Cas9 plasmid for GRB2 knockout.

[0052] First, 293T cells were cultured at 4 × 10 5The number of cells was inoculated in a 10 cm dish. The next day, after the cells adhered to the wall, the cell confluence was about 50%. After the cells were starved for 2 hours by replacing the serum-free DMEM medium, the CRISPR-Cas9 plasmid, pMD2.G and psPAX2 for GRB2 knockout were transfected into 293T cells at a ratio of 5 μg: 2.5 μg: 2.5 μg using jetPRIME (Polyplus, French) according to the manufacturer's protocol. After 8 hours, the complete medium was replaced, the viral supernatant was collected after 48 hours, and the second batch of viral supernatant was collected after another 24 hours. The collected viral supernatant is the lentiviral solution containing the gene knockout GRB2.

[0053] KYSE150 cells were plated at 3 × 10 5 The number of cells was inoculated in a 6-well plate. The next day, after the cells adhered to the wall, the lentiviral solution and complete medium were mixed at a ratio of 1:1 and added to the KYSE150 cells for infection for 8 hours. When the KYSE150 cells were in good condition, they were selected with puromycin. Only the control cells died, and the remaining cells were expanded and cultured. The protein of the amplified cell line was extracted and quantified by BCA method, and the knockout of GRB2 in the KYSE150 cell line was detected by WB.

[0054] The results are as follows Figure 7 As shown, it was shown that the GRB2 knockout cell line was successfully constructed and named KYSE150-GRB2 KO cell line.

[0055] Example 6

[0056] WT and GRB2-KO KYSE150 cells were seeded in six-well plates at a density of 1000 / well. After the cells adhered to the wall the next day, they were treated with HCPT-pt at concentrations of 0nM, 5nM, 10nM, and 20nM for 14 days and then fixed and stained for observation. The results are shown in Figure 8 As shown, Figure 8 A in the figure is a representative picture of clone formation experiment; Figure 8 B in the figure is the quantification of clone number by ImageJ. The results show that HCPT-pt has a concentration-dependent inhibitory effect on the clone formation of wild-type esophageal cancer cells, verifying that HCPT-pt has inhibitory activity against tumors with high expression of GRB2.

[0057] A scratch test cell model was constructed to observe the migration ability of WT and GRB2-KO esophageal cancer cells (KYSE150) in each well in a culture environment containing HCPT-pt. When the cells were 100% confluent, the tip of the gun was used to measure the ruler and scratch the back as perpendicularly as possible. After washing the cells, the medium was replaced with 2% low serum and treated with 0nM, 5nM, 10nM, and 20nM HCPT-pt, respectively. The healing of the scratch was observed after 48 hours. The results are shown in Fig. 9 As shown, Fig. 9 A in the figure is a representative picture of the scratch experiment; Fig. 9 B in the figure is a quantitative analysis of the healing rate of the scratch experiment, and the results verified that HCPT-pt has a significant inhibitory effect on the migration ability of esophageal cancer with high expression of GRB2.

[0058] A cell invasion model was constructed using culture chambers and matrix gel plates, and the invasion ability of WT and GRB2-KO esophageal cancer cells in each chamber was observed in the solvent control culture environment and the culture environment containing HCPT-pt, respectively. WT and GRB2 KO esophageal cancer cells were resuspended in serum-free culture medium, and 200 μL was added to the upper chamber (about 10,000 cells). The next day, the cells were treated with HCPT-pt at concentrations of 0nM, 50nM, 100nM, and 200nM, respectively. 600 μL of 20% serum culture medium was added to the lower chamber. After 48 hours, the cells were fixed with paraformaldehyde and stained with crystal violet, and the cell attachment at the bottom of the Transwell chamber was observed under a microscope. The results are shown in Fig.10 As shown, Fig.10 A in the figure is the Transwell assay to analyze the invasion ability of cells after HCPT treatment; Fig.10 B in the figure is the number of cells that penetrated the chamber quantified by ImageJ. The results verified that HCPT-pt had a significant inhibitory effect on the invasive ability of esophageal cancer with high expression of GRB2, indicating that HCPT-pt exerts its anti-cancer effect by targeting GRB2.

[0059] The above data show that HCPT-pt is a new small molecule inhibitor of GRB2, which can exert anti-tumor effects by targeting GRB2, and has far-reaching significance for the treatment of a wide range of tumor diseases.

Claims

1. A small molecule inhibitor of GRB2, characterized in that: Named as HCPT-pt, the structural formula is shown in Formula II, Formula II.

2. Use of the GRB2 small molecule inhibitor according to claim 1 in the preparation of anti-tumor drugs, wherein the tumor type is GRB2-positive esophageal cancer.

3. An anti-tumor drug, characterized in that: The active ingredient is the GRB2 small molecule inhibitor according to claim 1.

4. The method for preparing the GRB2 small molecule inhibitor according to claim 1, characterized in that: The phenolic hydroxyl group of hydroxycamptothecin is structurally modified, and 3-(2-bromoethyl)-3-(but-3-yn-1-yl)-3H-diaziridine is reacted with hydroxycamptothecin to obtain the GRB2 small molecule inhibitor.

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