A small molecule probe targeting mutant EGFR and a preparation method and application thereof

Abstract

The application discloses a small-molecule probe for targeting mutant EGFR, which comprises an EGFR-targeting binding group and a fluorescent group. The small-molecule probe can be used not only for imaging diagnosis of EGFR mutant tumors, but also for targeted therapy, and has an important clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a small-molecule probe targeting mutant EGFR and a preparation method and application thereof. BACKGROUND

[0002] Epidermal Growth Factor Receptor (EGFR) is a key member of the HER family (ErbB1), which as a transmembrane tyrosine kinase (EGFR-TK), triggers dimerization and intracellular kinase domain autophosphorylation by binding to ligands such as EGF and TGF-alpha, activates downstream signaling pathways (such as RAS / RAF / MEK / ERK, PI3K / Akt / mTOR), and regulates cell proliferation, survival and metastasis. In solid tumors such as non-small cell lung cancer (NSCLC), EGFR gene mutations (such as exon 19 deletion (19del), L858R point mutation and T790M drug resistance mutation) can cause abnormal activation of kinase activity and drive tumor progression.

[0003] Osimertinib is the first third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) approved by FDA, and is clinically approved for the treatment of non-small cell lung cancer patients with EGFR sensitive mutations (including L858R, 19del or superimposed T790M mutation). The main mechanism of action is that the osimertinib molecule contains an acrylamide group, which can form a covalent bond with the C797 cysteine residue in the EGFR kinase domain, achieve irreversible binding, inhibit its tyrosine kinase autophosphorylation, and block the activation of downstream key signaling pathways (such as RAS / RAF / MEK / ERK and PI3K / Akt / mTOR), thereby inhibiting tumor cell proliferation, survival and metastasis. However, after using osimertinib for about 9-13 months, EGFR is prone to third-generation TKI drug resistance mutations (such as C797S mutation), which causes the binding ability of EGFR to osimertinib to weaken and the sensitivity of the tumor to third-generation TKI to greatly decrease. This drug resistance mutation is often not discovered in time in clinical practice, leading to rapid deterioration of the disease. It is of great significance to improve the treatment effect of patients to discover the third-generation TKI drug resistance mutation of patients in time and implement intervention. The present application introduces a fluorescent probe based on the structure of osimertinib, which selectively binds to EGFR sensitive to osimertinib, can be used to judge whether the tumor EGFR is an osimertinib sensitive mutation (containing L858R, 19del, T790M mutation), and the molecule can also inhibit the activation of sensitive EGFR, achieving the function of killing tumor cells, and can be used as an EGFR sensitive mutation diagnosis and treatment integrated molecular probe. SUMMARY

[0004] An object of the present application is to provide a product capable of accurately diagnosing and treating non-small cell lung cancer with EGFR sensitive mutations in order to solve the above technical problems.

[0005] In order to achieve the above object, the present application provides a small molecule probe targeting mutant EGFR, which comprises an EGFR binding group and a fluorescent group.

[0006] Preferably, the chemical structure of the small molecule probe is as follows:

[0007] ,

[0008] wherein R is a fluorescent group.

[0009] Preferably, the fluorescent group is a near-infrared imaging group.

[0010] Preferably, the chemical structure of the small molecule probe is as follows:

[0011]

[0012] In another aspect, the present application also provides a method for preparing the small molecule probe, comprising the following steps:

[0013] Step 1. Mix and dissolve osimertinib and acrylic acid in water at a molar ratio of 1:6, stir and warm to 40℃, stir for 2 hours, after the reaction is completed, concentrate to dryness under reduced pressure, separate and purify the obtained residue by column chromatography, collect the eluate, concentrate to dryness under reduced pressure to obtain the crude product osimertinib acrylic acid derivative (OsiA), and then purify and separate it;

[0014] Step 2. Mix water-soluble cyanine 7 amino dye (Cy7-NH2), OsiA, EDCI and HOBt at a molar ratio of 1:1:1.5:1.5, dissolve in DMF, add DIEA to adjust the pH to 8.0, react at room temperature in the dark, separate the product after the reaction is completed, concentrate to dryness under reduced pressure, and obtain the purified small molecule probe OsiA-Cy7.

[0015] In another aspect, the present application also provides the use of the small molecule probe in the fluorescence detection of EGFR L858R mutation, EGFR19del mutation, or superimposed EGFR T790M mutant cells.

[0016] In another aspect, the present application also provides the use of the small molecule probe in the preparation of a product for diagnosing non-small cell lung cancer.

[0017] In another aspect, the present application also provides the use of the small molecule probe in the preparation of a drug for treating non-small cell lung cancer.

[0018] Preferably, the non-small cell lung cancer is EGFR mutant non-small cell lung cancer.

[0019] The small molecule probe OsiA-Cy7 of the present application can selectively target EGFR T790M / L858R and EGFR 19del mutants, and the probe is composed of an EGFR binding group and a fluorescent imaging group, wherein the EGFR binding group can specifically recognize and bind to the mutant cells, and the fluorescent imaging group can emit a fluorescent signal under excitation to realize real-time imaging monitoring of the EGFR mutant cells. In addition, the probe has cytotoxicity to the mutant cells, can effectively inhibit the mutant EGFR signal pathway, and thus can treat EGFR mutant non-small cell lung cancer. The small molecule probe of the present application can not only be used for imaging diagnosis of EGFR mutant tumors, but also can be used for targeted therapy, and has important clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The synthesis route of the small molecule probe OsiA-Cy7 of the present application is shown.

[0021] Figure 2 The UHPLC-MS / MS secondary spectrum of the small molecule probe OsiA-Cy7 of the present application is shown.

[0022] Figure 3 The absorption spectrum characterization result graph of the small molecule probe OsiA-Cy7 of the present application is shown.

[0023] Figure 4 The fluorescence spectrum graph of the small molecule probe OsiA-Cy7 of the present application is shown.

[0024] Figure 5 The flow result graph of the small molecule probe OsiA-Cy7 of the present application is shown.

[0025] Figure 6 The high-content microscope imaging result graph of the small molecule probe OsiA-Cy7 of the present application is shown.

[0026] Figure 7 The CCK8 experiment result graph of osimertinib and the small molecule probe OsiA-Cy7 of the present application on H1975 cells is shown.

[0027] Figure 8 The small molecule probe OsiA-Cy7 of the present application on A549, PC9, H1975 cell plate cloning experiment result graph is shown.

[0028] Figure 9Figure showing the results of Western Blot experiment of the small molecule probe OsiA-Cy7 on A549 cells.

[0029] Figure 10 Figure showing the results of Western Blot experiment of the small molecule probe OsiA-Cy7 on H1975 cells.

[0030] Figure 11 Figure showing the results of Western Blot experiment of the small molecule probe OsiA-Cy7 on PC9 cells.

[0031] Figure 12 Figure showing the results of Western Blot experiment of Osimertinib on PC9 cells. DETAILED DESCRIPTION

[0032] The application will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the application, and are not intended to limit the scope of the application.

[0033] Example 1: Synthesis and characterization of small molecule probe OsiA-Cy7

[0034] I. Synthesis of small molecule probe OsiA-Cy7

[0035] 1. Synthesis raw materials

[0036] Osimertinib (Osimertinib, Osimertinib, 1421373-65-0), water-soluble Cy7 (Sulfo-Cy7-NH2, Ruixibio, R-FR-062), acrylic acid (Macklin, A980638), N,N-dimethylamide (DMF, Macklin, N807505), 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDCI, Macklin, N808856), 1-hydroxybenzotriazole (HOBt, Macklin, H742556), N,N-diisopropyl ethylamine (DIEA, Macklin, N807281).

[0037] 2. Synthesis steps

[0038] (1) 1 g of Osimertinib and 1.4 g of acrylic acid (molar ratio 1:6) were mixed and dissolved in 10 mL of water, stirred and heated to 40°C, and stirred for 2 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure at 40°C, and the obtained residue was separated and purified by column chromatography (silica gel 200-300 mesh, eluent was dichloromethane and methanol, volume ratio of dichloromethane and methanol was 30:1), and the eluate was collected and concentrated to dryness under reduced pressure at 40°C to obtain Osimertinib acid OsiA crude product.

[0039] (2) The OsiA was purified by high performance liquid chromatography, acetonitrile (Merck, 1.00030.4008) was used as organic phase A, pure water was used as water phase B, and a C18 packing column (Cosmosil, 38024-01) was used for separation. The separation gradient was as follows: 0-2 min 10%B, 3-20 min 20%B, 20.5-30 min 30%B, 31-33 min 95%B, 34-37 min 10%B. The OsiA collection time was 26.0 min. The collected product was concentrated and dried under reduced pressure at 40°C to obtain purified OsiA.

[0040] (3) 15 mg of water-soluble Cy7, 10 mg of OsiA, 3.8 mg of EDCI, and 2.3 mg of HOBt were mixed in a molar ratio of 1:1:1.5:1.5, dissolved in 5 mL of DMF, 50 μL of DIEA was added, the pH was adjusted to 8.0, and the reaction was carried out at room temperature for 12 hours in the dark. After the reaction was completed, the product OsiA-Cy7 was obtained.

[0041] (4) The product was separated by high performance liquid chromatography, acetonitrile and pure water were used as mobile phase, the elution gradient was as follows: 0-3 min 80%B, 4-13 min 74%B, 14-21 min 70%B, 22-29 min 45%B, the preparation column type and mobile phase used were the same as those in step (2) above, and the OsiA-Cy7 collection time was 26.6 min. It was concentrated to dryness under reduced pressure at 45°C to obtain purified OsiA-Cy7.

[0042] The synthesis route of OsiA-Cy7 is shown in Figure 1 .

[0043] The purified small molecule probe OsiA-Cy7 was identified by UHPLC-MS / MS (Thermo Fisher, Q-Exactive Orbitrap MS), and the target product was successfully purified. Figure 2 The UHPLC-MS / MS secondary spectrum of the small molecule probe OsiA-Cy7 is shown.

[0044] II. Spectral characterization of the small molecule probe OsiA-Cy7

[0045] The optical characteristics of the synthesized and purified small molecule probe OsiA-Cy7 were characterized by an enzyme marker, and its near-infrared luminescence ability was verified.

[0046] 1. Experimental materials and instruments

[0047] Small molecule probe OsiA-Cy7 (synthesized by the method described in the above "1. Synthesis of small molecule probe OsiA-Cy7" section), PBS (Gibco, C10010500BT), 96-well plate (NEST, 701002), full-function microplate reader (Thermo Scientific Varioskan LUX).

[0048] 2. Experimental procedure

[0049] (1) Dilute the small molecule probe OsiA-Cy7 and the raw material water-soluble Cy7 to a concentration of 100 μM with PBS, and take 100 μL to add to the 96-well plate.

[0050] (2) Use the absorption spectrum module to set the scan range to 300-900 nm, step 1 nm, and perform absorption spectrum scanning on the blank control, water-soluble Cy7 and small molecule probe OsiA-Cy7.

[0051] (3) Use the fluorescence spectrum module to determine the fluorescence spectrum of the small molecule probe OsiA-Cy7. Set the excitation wavelength to 730 nm for the emission spectrum, and set the scan range to 750-840 nm; set the emission spectrum to 802 nm for the excitation spectrum determination, and set the scan range to 550-790 nm.

[0052] 3. Experimental results

[0053] Figure 3 The figure is the absorption spectrum characterization result of the small molecule probe OsiA-Cy7. As shown in Figure 3 , the absorption spectrum of the synthesized small molecule probe OsiA-Cy7 has obvious differences with the raw material water-soluble Cy7 in the range of 300-500 nm, and has the highest absorption peak at 750 nm, verifying that the small molecule probe OsiA-Cy7 retains the spectral characteristics of the raw material water-soluble Cy7.

[0054] Figure 4 The figure is the fluorescence spectrum of the small molecule probe OsiA-Cy7. As shown in Figure 4 , the maximum excitation wavelength of the small molecule probe OsiA-Cy7 is 747 nm, and the maximum emission wavelength is 776 nm.

[0055] Example 2: Verification of the targeting of the small molecule probe OsiA-Cy7 to EGFR mutant cells

[0056] One, flow cytometry verification of the targeting of the small molecule probe OsiA-Cy7 to different types of EGFR cell lines

[0057] The targeting of the small molecule probe OsiA-Cy7 to EGFR mutant cell lines (PC9, H1975) was verified by flow cytometry.

[0058] 1. Experimental grouping

[0059] A549 (wild-type EGFR WT); PC9 (EGFR 19del); H1975 (EGFR T790M / L858R).

[0060] 2. Experimental consumables and instruments

[0061] Small molecule probe OsiA-Cy7 (synthesized by the method described in the "Synthesis of small molecule probe OsiA-Cy7" section of Example 1), cell nucleus dye Hoechst 33342 (MCE, HY-15559), serum-free RPMI-1640 medium (Gibco, C11995501BT), phosphate buffer PBS (Gibco, C10010500BT), bovine serum albumin BSA (Roche, 10735086001), 0.25% w / v trypsin (Gibco, 25200072), 4% w / v paraformaldehyde fixing solution (Bi Yun Tian, P0099), 6-well plate (NEST, 703001); flow cytometer (Beckman Coulter Cytoflex LX), centrifuge (Thermo Biofuge Startos).

[0062] 3. Experimental procedure

[0063] (1) Plating: Take A549, PC9, and H1975 cells grown to 80% and count them after digestion using a bovine counting plate. Plate 2 x 10 5 cells per well in a 6-well plate. After growing for 48 hours, perform the experiment.

[0064] (2) Probe incubation: Dissolve Hoechst 33342 to 1 x and dilute small molecule probe OsiA-Cy7 to 5 μM using serum-free RPMI-1640 medium. Wash the cells twice with 1% BSA-PBS in the 6-well plate. Add 1 mL of the prepared small molecule probe OsiA-Cy7 incubation solution to each well and incubate at 37°C for 1 hour.

[0065] (3) Digestion: After incubation, aspirate the incubation solution and digest the cells using 500 μL of 0.25% trypsin for 2 min. Add 1 mL of 1% BSA-PBS to terminate the digestion and centrifuge at 1200 rpm for 3 min using a centrifuge. Discard the digestion solution and wash the cells twice with 1% BSA-PBS.

[0066] (4) Fixation: Cells were fixed with 1 mL 4% paraformaldehyde solution, and placed at room temperature for 20 min. After fixation, cells were centrifuged at 1200 rpm for 10 min, and the fixing solution was discarded. Cells were washed with 1% BSA-PBS for 3 times to remove free probes.

[0067] (5) Detection: Cells were resuspended with 500 μL 1% BSA-PBS, and 20000 single cells were detected by flow cytometry.

[0068] 4. Experimental results

[0069] Figure 5 The flow cytometry results of small molecule probe OsiA-Cy7 are shown in Figure 2. As shown in Figure 2, the flow cytometry fluorescence intensity of small molecule probe OsiA-Cy7 binding to A549 cells was significantly weaker than that of H1975 cells and PC9 cells, and the average fluorescence intensity was significantly statistically different (**p<0.01, ***p<0.001), indicating that small molecule probe OsiA-Cy7 can target and bind to EGFR sensitive mutant cell lines. Figure 5

[0070] Two, near-infrared imaging verification of small molecule probe OsiA-Cy7 targeting different types of EGFR cell lines

[0071] Different EGFR-expressing cell lines were incubated with small molecule probe OsiA-Cy7, and high-content microscopy was used for near-infrared imaging observation to verify the targeting of small molecule probe OsiA-Cy7 to EGFR sensitive mutant cell lines.

[0072] 1. Experimental grouping

[0073] A549 (wild-type EGFR WT); PC9 (EGFR 19del); H1975 (EGFR T790M / L858R).

[0074] 2. Experimental consumables and instruments

[0075] ​Small molecule probe OsiA-Cy7 (synthesized by the method described in the "Part I, Synthesis of small molecule probe OsiA-Cy7" section of Example 1), Hoechst 33342 (MCE, HY-15559), RPMI-1640 (Gibco, C11995501BT), PBS (Gibco, C10010500BT), BSA (Roche, 10735086001), 0.25% trypsin (Gibco, 25200072), 4% paraformaldehyde fixing solution (Bi Yun Tian, P0099), 96-well glass bottom culture plate (Cellvis, P96-1.5H-N); high content microscope (ImageXpress® Confocal HT.ai), centrifuge (Thermo Biofuge Startos).

[0076] 3. Experimental procedure

[0077] (1) Cell plating: A549, H1975, PC9 cells growing to 80-90% were digested from the culture dish using 0.25% trypsin (EDTA) and centrifuged to collect (1200 rpm for 3 min), and the cells were resuspended in 5 mL of RPMI-1640 medium containing 10% FBS, mixed well, and then 10 μL was added to a clean bovine counting plate to fill the pool, and left to stand for 5 min. After the cells settled, the bovine counting plate was counted using the four large squares in the corners, and the number of cells per μL was calculated: total number of cells in four large squares / 4*10 = number of cells per μL. The cells were plated at a density of 5000 cells per well, and after plating, the 96-well plate was placed in a 37°C incubator for 24 hours or more.

[0078] (2) Probe incubation: After the cells adhered, they were blocked with 1% BSA-PBS solution for 30 min. 5 mM OsiA-Cy7 and 5 mM Cy7 probes were diluted to working concentration using serum-free medium. After blocking, the blocking solution was removed, and the corresponding incubation solution was added to each well and incubated at 37°C for 1 hour. After incubation, the probe solution was removed, and the 96-well plate was washed with PBST twice.

[0079] (3) Fixation: 200 μL of 4% paraformaldehyde fixing solution was added to each well, and fixed at room temperature for 20 min. After fixation, the wells were washed 5 times with PBST.

[0080] (4) Imaging: Confocal mode imaging was performed using a high content microscope, and Cy7 and DAPI channels were used for photography.

[0081] 4. Experimental results

[0082] Figure 6 The small molecule probe OsiA-Cy7 high content microscope imaging results are shown in the figure. AsFigure 6 As shown in the figure, the small molecule probe OsiA-Cy7 has less retention on A549 cells, and has strong near-infrared fluorescence signal on H1975 and PC9 cells. Through comparison and analysis of the average fluorescence intensity of the cells, it was found that the average fluorescence intensity of H1975 and PC9 cells had a statistically significant difference compared with A549 cells (p<0.001). The above experimental results show that the small molecule probe OsiA-Cy7 has good selectivity for EGFR sensitive mutation and drug-resistant mutation cell lines (PC9, H1975).

[0083] Example 3: Verification of the therapeutic effect of small molecule probe OsiA-Cy7 on EGFR sensitive mutation cell lines

[0084] I. Cytotoxicity experiment

[0085] The cytotoxicity experiment was used to verify that the small molecule probe OsiA-Cy7 has similar EGFR mutant cell line cell viability inhibition effect as the drug osimertinib, and to prove its potential therapeutic ability for EGFR mutant non-small cell lung cancer.

[0086] 1. Experimental grouping

[0087] Cell line: H1975 (EGFR T790M / L858R);

[0088] Drug: Osimertinib, small molecule probe OsiA-Cy7;

[0089] Concentration gradient (nM): 0, 1, 10, 25, 50, 125, 250, 500, 1000.

[0090] 2. Experimental consumables and instruments

[0091] Small molecule probe OsiA-Cy7 (synthesized by the method described in the "Synthesis of small molecule probe OsiA-Cy7" section of Example 1), Osimertinib (Widely reagent, 1421373-65-0), RPMI-1640 (Gibco, C11995501BT), PBS (Gibco, C10010500BT), FBS (Excell, FSP500), 0.25% trypsin (Gibco, 25200072), 4% paraformaldehyde fixing solution (Bi Yun Tian, P0099), 96-well plate (NEST, 701002), CCK8 kit (biosharp, BS350D); Full-function microplate detector (THermo Scientific Varioskan LUX).

[0092] 3. Experimental procedure

[0093] (1) Cell plating: H1975 cells that have grown to 80-90% were digested from the culture dish with 0.25% trypsin (EDTA) and collected by centrifugation (1200 rpm for 3 min). The cells were resuspended in 5 mL of 10% FBS RPMI-1640, mixed well, and 10 μL was added to the cleaned Nieuws-Bauer counting plate. The plate was then allowed to stand for 5 min. After cell sedimentation, the cells were counted using the four large squares of the Nieuws-Bauer counting plate. The number of cells per μL was calculated as: total number of cells in the four large squares / 4 * 10 = number of cells per μL. The plate was calculated to be 5000 cells per well. After plating, the 96-well plate was placed in a 37℃ incubator for at least 24 hours.

[0094] (2) Drug incubation: Osimertinib and small molecule probe OsiA-Cy7 were prepared in medium at a concentration of 0-1000 nM and added to fully adherent H1975 cells, 150 μL per well, and cultured at 37°C for 72 hours.

[0095] (3) CCK8 detection: After incubation, aspirate the culture medium from each well. Add culture medium containing CCK-8 (1 / 10 of the cell culture medium volume) and incubate at 37°C for 2 hours. After the reaction, use a full-function microplate reader to detect the cells at a wavelength of 450 nm. Graphpad is then used to analyze the results and calculate the half-inhibition concentration (IC50). 50 ).

[0096] 4. Experimental Results

[0097] Figure 7 The image shows the results of CCK8 assays on H1975 cells using osimertinib and the small molecule probe OsiA-Cy7. Figure 7 As shown, osimertinib has an effect on the IC50 response of H1975 cells. 50 The IC50 value of OsiA-Cy7 in H1975 cells was 10.93 nM. 50 The concentration was 9.983 nM, and the inhibitory effects of the two drugs were similar, demonstrating that OsiA-Cy7 has a growth inhibitory effect on EGFR-sensitive mutant cell lines and has the potential to treat EGFR-sensitive mutant non-small cell lung cancer.

[0098] II. Plate Cloning Experiment

[0099] The growth-inhibiting effect of the small molecule probe OsiA-Cy7 on different types of EGFR mutant cell lines was verified by plate cloning experiments, demonstrating its ability to target EGFR mutant non-small cell lung cancer.

[0100] 1. Experimental Grouping

[0101] Cell lines: A549 (wild type EGFR WT), PC9 (EGFR 19del), H1975 (EGFR T790M / L858R);

[0102] Drug: small molecule probe OsiA-Cy7;

[0103] Concentration gradient (nM): 0, 1, 10, 100, 1000.

[0104] 2. Experimental consumables and instruments

[0105] Small molecule probe OsiA-Cy7 (synthesized by the method described in the "Synthesis of small molecule probe OsiA-Cy7" section of Example 1), RPMI-1640 (Gibco, C11995501BT), PBS (Gibco, C10010500BT), FBS (Excell, FSP500), 0.25% trypsin (Gibco, 25200072), 6-well plate (NEST, 703001), crystal violet dye (BKMAMLAB, 110703008).

[0106] 3. Experimental procedure

[0107] (1) Cell plating: Use 0.25% trypsin (EDTA) to digest A549, PC9, H1975 cells growing to 80-90% from the culture dish and centrifuge to collect (1200 rpm for 3 min), resuspend the cells with 5 mL 10% FBS RPMI-1640, mix well and take 10 μL to fill the pool of a clean bovine counting plate, stand for 5 min. After the cells settle, use the four corners of the bovine counting plate to count the number of cells in each μL: total number of cells in four large squares / 4*10 = number of cells per μL. Plate at 1000 cells per well, and place the 6-well plate in a 37°C incubator for 24 hours or more after plating.

[0108] (2) Drug culture: Dilute the small molecule probe OsiA-Cy7 to 1-1000 nM with complete culture medium, and add the drug-containing medium to the 6-well plate, 37°C culture for 10 days, and change the medium every three days.

[0109] (3) Fixation and staining: remove the culture medium, wash the cells with PBS, fix the cells with 4% paraformaldehyde for 20 min; remove the paraformaldehyde and stain with 0.2% crystal violet for 5 min; wash the cells with tap water, air dry, and take pictures with a camera.

[0110] 4. Experimental results

[0111] Figure 8The image shows the results of a plate cloning experiment using the small molecule probe OsiA-Cy7 on A549, PC9, and H1975 cells. Figure 8 As shown, the small molecule probe OsiA-Cy7 had no significant inhibitory effect on A549 cells at concentrations of 0-100 nM, but exhibited significant growth inhibition in EGFR-mutant PC9 and H1975 cells at concentrations of 1-10 nM. These results demonstrate that the small molecule probe OsiA-Cy7 has potential targeted therapeutic effects on EGFR-mutant cell lines.

[0112] III. Western blot verification

[0113] Western blotting experiments were used to verify the inhibitory effect of the small molecule probe OsiA-Cy7 on EGFR / Akt phosphorylation in different types of EGFR mutant cell lines, demonstrating that it can block the activation of the downstream PI3K / Akt / mTOR pathway by inhibiting EGFR phosphorylation, thereby achieving targeted therapy for EGFR mutant non-small cell lung cancer.

[0114] 1. Experimental Grouping

[0115] Cell lines: A549 (wild-type EGFR WT), PC9 (EGFR 19del), H1975 (EGFR T790M / L858R);

[0116] Drugs: Osimertinib, small molecule probe OsiA-Cy7, EGF (epidermal growth factor);

[0117] OsiA-Cy7 concentration gradient (nM): 0, 1, 10, 100, 1000.

[0118] 2. Experimental Consumables and Instruments

[0119] OsiA-Cy7 (synthesized by the method described in the "1. Synthesis of small molecule probe OsiA-Cy7" section of Example 1), RPMI-1640 (Gibco, C11995501BT), PBS (Gibco, C10010500BT), FBS (Excell, FSP500), 0.25% trypsin (Gibco, 25200072), 6-well plate (NEST, 703001), EGF (Peprotech, AF-100-15), One-Step Gel Preparation Kit (10%) (Fugene, FD341-100), 10% APS (Source Leaf Biotech, R21206), 5x Loading buffer (Fugene, FD006), RIPA lysis buffer (Fugene, FD009), protease / phosphatase inhibitor (Thermo, 78447), PMSF (Fugene, FD0100), Tris (Macklin, T819512), Glycine (Macklin, G800883), Methanol (Guangzhou brand, HB05), pre-stained protein marker (Thermo, 26620), Tween 20 (Fugene, FD0020), BSA (Roche, 10735086001), Anti-EGFR (CST, 4267), Anti-pEGFR (Y1068) (CST, 3777), Anti-Akt (CST, 9272), Anti-pAkt (S473) (4060), Anti-β-Actin (CST, 4970), Rabbit secondary antibody (Fugene, FD0115), primary antibody diluent (Fugene, FD0040), PVDF membrane (Merck, IPVH00010), ECL developing solution (Fugene, FD8020).

[0120] 3. Experimental procedure

[0121] (1) Cell plating: Use 0.25% trypsin (EDTA) to digest A549, PC9, H1975 cells growing to 80-90% from the culture dish and collect by centrifugation (1200 rpm, 3 min), resuspend the cells with 5 mL 10% FBS RPMI-1640, mix well and take 10 μL to the washed bovine counting chamber for cell counting, and stand for 5 min. After the cells settle, use the four corners of the bovine counting chamber to count the number of cells in each large square: total number of cells in four large squares / 4*10 = number of cells per μL. Calculate the plating with 5x10 5 cells per well, and after plating, place the 6-well plate in a 37°C incubator for more than 24 hours.

[0122] (2) Incubate the wells except the blank control with the medium containing 10 ng / mL EGF. Dilute the small molecule probe OsiA-Cy7 to the corresponding concentration with the prepared medium, and incubate the medium containing the drug in each well at 37°C for 24 hours.

[0123] (3) After the incubation is completed, protein extraction is performed. Prepare RIPA lysis solution, ice tray, pre-cooled PBS, etc. in advance. Take out the 6-well plate, wash the cells with pre-cooled PBS for 2 times, add 50-150 μL of RIPA lysis solution according to the cell density, scrape the cells with a scraper, collect them into an EP tube, and stand on ice for 30 min. Pre-cool the high-speed centrifuge.

[0124] (4) After standing and lysing, ultrasonic for 3 min in ice water bath, use high-speed centrifuge at 13000 rpm for 10 min to collect the supernatant. Add loading buffer to 1x concentration, denature the protein at 37°C metal bath for 30 min, and immediately place it on ice after denaturation. Divide and store for use.

[0125] (5) Prepare SDS-PAGE gel, electrophoresis solution, and membrane transfer solution according to the instructions.

[0126] (6) According to the protein extraction density, determine the loading amount, and add the sample incubated with loading buffer after denaturation to the loading slot. Use 1x loading buffer to make up the loading amount, add electrophoresis solution, and run the sample at 80V for 30 min and 120V for 55 min.

[0127] (7) Prepare 1x membrane transfer solution in advance and pre-cool it in the 4°C refrigerator. Activate the cut PVDF membrane with methylase. After the gel is run, cut off the excess components, assemble the membrane transfer device according to the sandwich method, and make sure that there are no bubbles between the membrane and the gel. Transfer the membrane at 260mA constant current for 150 min. After the transfer is completed, take out the PVDF membrane and wash it with TBST once. At this time, take a photo of the whole membrane with a camera.

[0128] (8) Block the PVDF membrane with 5% BSA-TBST at room temperature for 1.5 hours.

[0129] (9) Take out the corresponding primary antibody, cut the membrane according to the position of the target protein, and add the corresponding primary antibody after cutting. Incubate at 4°C overnight.

[0130] (10) After incubation with the primary antibody, wash the membrane with 1x TBST for 3 times, each for 10 min. After washing, add the corresponding secondary antibody and incubate on a shaker at room temperature for 1.5 hours.

[0131] (11) After the secondary antibody incubation is completed, wash the membrane three times with 1×TBST, 10 min each time. After washing the membrane, enter the darkroom, turn on the developer and pre-cool the camera. Prepare ECL developer solution with a ratio of A solution:B solution = 1:1. Start development after the instrument is ready.

[0132] 4. Experimental Results

[0133] Figure 9 to Figure 11 The image shows the results of Western blotting experiments on A549, PC9, and H1975 cells using the small molecule probe OsiA-Cy7. Figure 9 As shown, the small molecule probe OsiA-Cy7 had no significant inhibitory effect on EGFR phosphorylation in A549 cells at concentrations of 0-100 nM, while... Figure 10 and Figure 11 As shown, PC9 and H1975 cells with EGFR mutations exhibited significant inhibition of EGFR and Akt phosphorylation at 10 nM. Simultaneously, PC9 cells were incubated with osimertinib, as... Figure 12 As shown, the same phosphorylation inhibition effect as the small molecule probe OsiA-Cy7 was also observed. These results demonstrate that the small molecule probe OsiA-Cy7 has a targeted therapeutic effect on EGFR mutant cell lines and can effectively inhibit the activation of the PI3K / Akt / mTOR pathway.

Claims

1. A small molecule probe targeting mutant EGFR, characterized in that, The small molecule probe includes an EGFR binding group and a fluorescent group; The chemical structural formula of the small molecule probe is as follows: 。 2. A method for preparing the small molecule probe as described in claim 1, characterized in that... Includes the following steps: Step 1. Osimertinib and acrylic acid were mixed and dissolved in water at a molar ratio of 1:

6. The mixture was stirred and heated to 40°C. The mixture was stirred and reacted for 2 hours. After the reaction was completed, the mixture was concentrated to dryness under reduced pressure. The residue was separated and purified by column chromatography. The eluent was collected and concentrated to dryness under reduced pressure to obtain crude product OsiA, which was then purified and separated. Step 2. Mix water-soluble Cy7, OsiA, EDCI and HOBt in a molar ratio of 1:1:1.5:1.5, dissolve in DMF, add DIEA to adjust pH to 8.0, react at room temperature in the dark, after the reaction is complete, separate the product, concentrate under reduced pressure to dryness, and obtain the purified small molecule probe.

3. The use of the small molecule probe as described in claim 1 in the preparation of products for fluorescent detection of EGFR L858R mutant, EGFR 19del mutant, or superimposed EGFR T790M mutant cells.

4. The use of the small molecule probe as described in claim 1 in the preparation of products for diagnosing non-small cell lung cancer.

5. The use of the small molecule probe as described in claim 1 in the preparation of a medicament for treating non-small cell lung cancer.

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