Use of dye molecules in detecting tyrosine phosphorylated peptides and monitoring protein kinase activity

Abstract

This invention provides a fluorescent probe for label-free, multifunctional, low-cost, and high-throughput real-time monitoring of tyrosine phosphorylation. Our designed fluorescent molecule specifically binds to copper ions in aqueous solution, leading to fluorescence quenching. Adding a phosphorylated substrate peptide with tyrosine residues to the quenched system elicits a dramatic change in fluorescence, with the solution color changing from purple to red. However, no significant fluorescence change is observed upon adding non-phosphorylated substrate peptides or other phosphate-related ions. This on-off fluorescence response enables real-time monitoring of tyrosine phosphorylation, with a detection limit of 100 nM. This method can also be applied to the rapid screening of PKA kinase inhibitors and to the detection of serine, threonine, and phosphorylated peptides at mono- and diphosphorylated sites. The greatest advantage of this fluorescence spectroscopy method is that it does not require antibodies or radioactive isotopes, has simple operating procedures, and is highly suitable for the real-time detection of protein tyrosine phosphatase activity.

Description

Technical Field

[0001] This invention belongs to the field of analytical technology, and specifically relates to a label-free, multifunctional, low-cost, and high-throughput method for monitoring tyrosine phosphorylation. Background Technology

[0002] Protein phosphorylation is one of the most fundamental, common, and important biological processes in metabolism. It controls many intracellular signal transductions and regulates various cellular activities, including cell growth, differentiation, and apoptosis. Strong evidence suggests that abnormal protein kinase activity disrupts the protein phosphorylation network, leading to various human diseases. For example, cancer cells are often observed to have excessively active protein phosphorylation levels due to gene mutations or other mechanisms. As of 2020, the U.S. Food and Drug Administration (FDA) had approved approximately 52 small molecule inhibitors of protein kinases. These drugs are primarily multi-target receptor tyrosine kinase (RTK) inhibitors approved for the treatment of cancer.

[0003] Developing targeted cancer drugs often involves monitoring protein kinase activity, with mainstream methods including the use of radioactivity. 32 Methods for labeling ATP with P and immunoassays using specific antibodies against phosphorylated residues have made significant progress in assessing protein kinase activity. However, the use of radioactivity... 32 The detection of PKA generates a large amount of radioactive waste, causing serious environmental pollution. Furthermore, the analysis of radioactive compounds requires prolonged exposure to a radioactive environment, which seriously endangers human health. Antibody-based immunoassays typically require expensive and specific antibodies, and the isolation and purification of these antibodies involves complex processes. Therefore, there is an urgent need to develop new methods for monitoring tyrosine phosphorylation. Summary of the Invention

[0004] The purpose of this invention is to provide a novel method for the detection of tyrosine-phosphorylated peptides and the monitoring of protein kinase activity. This probe can be used for the qualitative and quantitative detection of tyrosine-phosphorylated peptides, and can also be used as a monitoring method for protein kinase activity. Compared with traditional enzyme-linked immunosorbent assays (ELISA) and mass spectrometry, this probe offers advantages such as simple operation, low cost, and no labeling required when detecting tyrosine-phosphorylated peptides. It is highly suitable for the detection and analysis of tyrosine-phosphorylated peptides.

[0005] The detection principle of this probe is based on the specific binding of the dye molecule H-HPMO to copper ions in aqueous solution, which leads to the quenching of H-HPMO fluorescence. When phosphorylated peptides or substrate peptides are present in the solution and phosphorylated by protein kinases, the phosphate groups on the peptides will competitively subtract copper ions, resulting in the recovery of H-HPMO fluorescence. This on-off fluorescence response allows for the detection of tyrosine-phosphorylated peptides and the monitoring of protein kinase activity. In kinase inhibitor screening experiments, if a candidate inhibitor can inhibit protein kinase-catalyzed substrate phosphorylation, the tyrosine groups on the substrate peptides cannot generate phosphate groups. Therefore, the dye molecule H-HPMO continues to react with Cu... 2+ When the binding is tight, the fluorescence is quenched, and the microplate reader displays the initial fluorescence value. Therefore, the magnitude of the fluorescence value can be used to determine whether a candidate inhibitor can inhibit the phosphorylation of substrates by protein kinases. Based on this principle, it can be used for the detection of phosphorylated peptides, the monitoring of protein kinase activity, and the preliminary screening of kinase inhibitors.

[0006] The technical solution of this invention is:

[0007] Fluorescent probes are used for the detection of tyrosine-phosphorylated peptides and the monitoring of protein kinase activity.

[0008] The probe molecule H-HPMO was dissolved in HEPES (50 mmol·L⁻¹). –1 In a buffer solution (pH = 7.3), copper nitrate was added and mixed thoroughly. Then, various prepared phosphorylated peptide solutions were added. The fluorescence response of the probe molecules to different phosphorylated peptides was detected using a fluorescence spectrometer. The solution containing the probe molecules H-HPMO–Cu was then added. 2+ Different concentrations of tyrosine-phosphorylated peptides were added to the test solution, and fluorescence values ​​at specific wavelengths were recorded. A standard curve was established based on the relationship between concentration and fluorescence intensity ratio. Protein kinase was added to the test solution, and the process of protein kinase catalyzing the formation of phosphorylated peptides from substrate peptides was monitored using an ELISA reader. Kinase inhibitors were added to the kinase reaction solution, and the process of inhibitors inhibiting protein kinase catalyzing the formation of phosphorylated peptides from substrate peptides was monitored using an ELISA reader. Common ion-pair probe molecules H-HPMO–Cu were investigated by adding different ions to the test solution. 2+ The effect of fluorescence properties.

[0009] The phosphorylated peptide solution includes monotyrosine phosphorylated peptide, dityrosine phosphorylated peptide, and trityrosine phosphorylated peptide; the protein kinase is tyrosine protein kinase c-Abl; the ions are sodium ions, magnesium ions, calcium ions, nitrate ions, carbonate ions, sulfate ions, isothiocyanate ions, and adenine nucleoside triphosphate (ATP); in all the above tests, the excitation wavelength of the fluorescence spectrometer is set to 500 nm, and the excitation wavelength of the enzyme-linked immunosorbent assay (ELISA) reader is set to 560 nm.

[0010] Step 1: Synthesize the probe molecule H-HPMO

[0011] The molecule was synthesized by condensing the aldehyde group on the intermediate HPMO with a heterocyclic quaternary ammonium salt containing an active methyl group under anhydrous conditions, followed by recrystallization with methanol. The solid was then repeatedly washed with methanol to obtain the final product.

[0012] Step 2, H-HPMO–Cu 2+ Preparation of test solution

[0013] The dye H-HPMO synthesized in step 1 was added to a HEPES buffer solution and sonicated until completely dissolved. Then, an equimolar amount of copper nitrate was added, and the mixture was stirred at room temperature to obtain H-HPMO–Cu. 2+ Test solution.

[0014] Step 3: Detect standard tyrosine phosphorylated peptides.

[0015] The H-HPMO–Cu prepared in step 2 was tested using an excitation wavelength of 500 nm. 2+ The fluorescence emission spectrum of the test solution is recorded as the initial fluorescence spectrum. Then, mono- or di-tyrosine phosphorylated peptides and their corresponding non-phosphorylated peptides are added separately, and fluorescence spectroscopy tests are performed, recording the corresponding fluorescence emission spectra. The presence of the probe H-HPMO–Cu is determined by comparing the fluorescence spectrum of the test solution with the initial fluorescence spectrum. 2+ It can be used to detect tyrosine-phosphorylated peptides.

[0016] Step 4: Quantitative analysis of tyrosine-phosphorylated peptides;

[0017] The H-HPMO–Cu prepared in step 2 was tested using an excitation wavelength of 500 nm. 2+ The fluorescence intensity value of the test solution at 598 nm is recorded as the initial fluorescence value.

[0018] Prepare standard solutions of tyrosine phosphorylated peptides at different concentrations, and then add the above solutions to a solution containing H-HPMO–Cu prepared in step 2. 2+ The fluorescence intensity value at 598 nm for each concentration was recorded in the test solution, and a standard curve was established by the relationship between the concentration of tyrosine phosphorylated peptide and the fluorescence intensity ratio.

[0019] Add an unknown concentration of tyrosine phosphorylated peptide to H-HPMO–Cu prepared in step 2 2+ The fluorescence intensity value at 598 nm was recorded in the test solution, and the corresponding concentration was calculated by substituting this value into the standard curve.

[0020] Step 5, monitoring protein kinase activity

[0021] To prepare the kinase reaction mixture, add the standard stock solution of the substrate peptide EE-13 to Tris-HCl buffer, and then add various salt solutions including ATP. 2- MgCl2 and NaCl. First group of experiments: 200 μL of the above reaction mixture was added to a black 96-well plate, followed by the H-HPMO–Cu prepared in step 2. 2+ The test solution was tested, and the background fluorescence value at 598 nm was recorded between 0 and 60 min.

[0022] In the second group of experiments, c-Abl protein kinase stock solution was added to the above reaction mixture, and after incubation at 37°C for 5 min, it was added to a black 96-well plate. Simultaneously, H-HPMO–Cu prepared in step 2 was rapidly added. 2+ The test solution was tested, and the change in fluorescence intensity at 598 nm was recorded between 0 and 60 min.

[0023] Step 6, kinase inhibitor screening;

[0024] Various candidate protein kinase inhibitors were added to the reaction mixture described in step 5, and the fluorescence intensity was recorded at 60 min under the same conditions as the second group of experiments in step 5.

[0025] The inhibitory effect of a candidate kinase inhibitor can be determined by the fluorescence values ​​at 598 nm of the test solution with and without protein kinase inhibitor.

[0026] In step (1) of the above method, the reaction temperature is set to 65℃, the reaction time is 12 to 18 hours, ultra-dry methanol is used as the reaction solvent, methanol is used as the solvent for recrystallization, and the molar ratio of the reaction raw materials is 1:1.

[0027] In step (2) of the above method, the concentration of the dye H-HPMO can be 3–300 μmol·L. –1 Specifically, it can be 33 μmol·L –1 The ultrasonic oscillation time can be 2-10 minutes, specifically 5 minutes.

[0028] In step (3) of the above method, H-HPMO–Cu 2+ The test solution volume can be 2–4 mL, specifically 3 mL; the standard phosphorylated peptide concentration is 0.005 mol, and the volume can be 20–80 μL, specifically 60 μL. The test temperature is room temperature, and the buffer solution can be 25 mmol·L⁻¹. –1 Tris, 50 mmol·L –1 Tris, 25 mmol·L –1 HEPES, 50 mmol·L –1 HEPES, specifically 50 mmol·L–1 HEPES caching solution.

[0029] In step (4) above, the concentration of tyrosine phosphorylated peptide is 29 μmol·L⁻¹. –1 33 μmol·L –1 37.5 μmol·L –1 45 μmol·L –1 54 μmol·L –1 58 μmol·L –1 62 μmol·L –1 66 μmol·L –1 Probe H-HPMO–Cu 2+ The concentration is 33 μmol·L –1 The detection system is 50 mmol·L⁻¹ –1 HEPES buffer solution, with fluorescence intensity value selected at 598nm.

[0030] The concentration of H-HPMO in step (5) above is 33 μmol·L. –1 The volume was 3 mL. The test was performed using a microplate reader, with adenosine triphosphate (ATP) volume of 15 μL (0.1 mol·L⁻¹). –1 ), magnesium chloride volume 300 μL (0.1 mol·L⁻¹) –1 ), Sodium chloride volume 300 μL (0.1 mol·L⁻¹) –1 The substrate peptide EE-13 was 120 μL (0.005 mol·L⁻¹). –1 ), kinase c-Abl 10μL (0.24μg / μL), Tris-HCl buffer volume 2255μL.

[0031] The program was set to use a dual-track vibration mode (medium speed) to ensure uniform mixing of the reaction solution, and the final concentration of c-Abl kinase was 100 nmol·L⁻¹. –1

[0032] The concentration of the candidate inhibitor in step (6) of the kinase inhibitor screening experiment above is 5 μmol·L⁻¹. –1 The parameters were set to incubate at 37℃ for 60 min, using linear dual-track vibration mode (medium speed). The sample volume in the well plate could be 200-300 μL, but the actual volume was 200 μL.

[0033] This invention provides a fluorescent probe for label-free, multifunctional, low-cost, and high-throughput real-time monitoring of tyrosine phosphorylation. The fluorescent molecule specifically binds to copper ions in aqueous solution, causing fluorescence quenching. Adding a phosphorylated substrate peptide with tyrosine residues to the quenched system elicits a dramatic change in fluorescence, with the solution color changing from purple to red. However, no significant fluorescence change is observed upon adding non-phosphorylated substrate peptides or other phosphate-related ions. This on-off fluorescence response enables real-time monitoring of tyrosine phosphorylation, with a detection limit of 100 nM. This method can also be applied to the rapid screening of protein kinase inhibitors and to the detection of serine, threonine, and phosphorylated peptides at mono- and diphosphorylated sites. The greatest advantage of this fluorescence spectroscopy method is that it does not require antibodies or radioactive isotopes, has simple operation steps, and is highly suitable for the real-time detection of protein tyrosine phosphatase activity.

[0034] The beneficial effects of this invention are: based on H-HPMO–Cu 2+ This invention enables the detection of standard phosphorylated peptides, real-time monitoring of tyrosine phosphorylation, and preliminary screening of protein kinase inhibitors. Compared with traditional detection methods, this invention does not require special signal molecule markers, is simple to operate, low in cost, high-throughput, and does not require complex instruments, making it valuable for targeted drug development. Attached Figure Description

[0035] Figure 1 The synthetic route for the probe H-HPMO is shown.

[0036] Figure 2 The data are the proton NMR characterization data of the probe H-HPMO.

[0037] Figure 3 The data are the carbon NMR characterization data of the probe H-HPMO.

[0038] Figure 4 High-resolution mass spectrometry characterization data for probe H-HPMO.

[0039] Figure 5 The infrared spectral characterization data are for the probe H-HPMO.

[0040] Figure 6 H-HPMO–Cu 2+ In the test solution, mono-, di-, or tri-tyrosine phosphorylated peptides and their corresponding non-phosphorylated peptides are added for fluorescence spectroscopy testing.

[0041] Figure 7 For H-HPMO–Cu 2+ Fluorescence spectra of 2pY, a bistyrosine phosphorylated peptide, were obtained by adding different concentrations of the test solution. The excitation wavelength was 500 nm, and the system volume was 50 mmol·L⁻¹. –1HEPES-NaOH buffer.

[0042] Figure 8 This is a fitting plot of the concentration of the 2pY bistyrosine phosphorylated peptide versus the fluorescence intensity ratio.

[0043] Figure 9 This is a standard curve showing the change in tyrosine phosphorylated peptide concentration with fluorescence intensity.

[0044] Figure 10 It incorporates common metal cations, anions, adenine nucleoside triphosphate (ATP), adenosine diphosphate (ADP) probes H-HPMO–Cu 2+ Fluorescence spectrum.

[0045] Figure 11 It is H-HPMO–Cu 2+ A graph showing the change in fluorescence intensity over time after the substance is added to the protein kinase reaction solution.

[0046] Figure 12 The fluorescence values ​​of different candidate inhibitors added to the protein kinase reaction solution are shown in the figure. The excitation wavelength is 560 nm, the emission wavelength is 598 nm, and the time point is 60 min. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. The raw materials used in the examples were: 1,3-dichloropropanone (99% purity), 2-hydroxybenzamide (99% purity), iodoethane (99% purity), potassium carbonate (K2CO3, 97% purity), potassium iodide (KI, 99% purity), 4-(diethylamino)-2-hydroxybenzaldehyde (99% purity), and 2,3,3'-trimethylindole (99% purity) purchased from Aladdin (Aladdin Biochemical Technology Co., Ltd., Shanghai, China); a series of tyrosine (Tyr) phosphorylated peptides (PPs) (1pY, 2pY, 3pY) and non-phosphorylated peptides (NMP) (purity >99.5%) with mono, di, or trityrosine groups purchased from ChinaPeptides (Shanghai, China); and c-Abl protein kinase substrate peptide EAIYAAPAAYIAE (EE-13) purchased from Sino Biological Company (Beijing, China) uses reagents or instruments whose manufacturers are not specified; these are all commercially available, standard products.

[0048] Example 1. Synthesis process of probe molecule H-HPMO.

[0049] The probe H-HPMO was synthesized through a three-step reaction, as shown in Figure 1.

[0050] 1) Synthesis: 1-Ethyl-2,3,3-trimethylindoleniniμm iodine (also known as hemicyanin)

[0051] 2,3,3-Trimethylindole (1 mL, 6.2 mmol) and iodoethane (1.45 g, 9.3 mmol) were mixed in 20 mL of anhydrous acetonitrile (CH3CN) solution and heated under reflux for 12 hours. The reaction mixture was then cooled to room temperature, and a pale red precipitate precipitated. The precipitate was filtered and washed three times with 20 mL of acetonitrile (3 × 20 mL). Finally, pink crystals (1.50 g, 90% yield) were obtained by vacuum drying.

[0052] 2) Synthesis: 2-(2'-hydroxyphenyl)-4-chloromethyloxazole

[0053] The synthesis of 2-(2'-hydroxyphenyl)-4-chloromethyloxazole is simple, solvent-free, and yields a high amount. First, 2-hydroxybenzamide (0.68 g, 5 mmol) and 1,3-dichloropropanone (1.26 g, 10 mmol) were added to a round-bottom flask. The reaction mixture was then stirred at 150 °C for 6 hours. After the reaction was complete, the mixture was cooled to room temperature, and saturated brine (200 mL) was added. The mixture was extracted three times with 200 mL of EtOAc (3 × 200 mL). The combined extracts were washed with brine (400 mL) and dried over anhydrous Na₂SO₄. The mixture was filtered, and the solvent was evaporated to dryness. The crude residue was purified by silica gel (100-200 mesh) column chromatography, eluting with ethyl acetate / petroleum ether (v / v, 1:9) to give the desired product (0.93 g, yield: 90%).

[0054] 3) Synthesis: 3H-Indoliμm,2-[2-[4-(diethylamino)-2-hydroxyphenyl]et enyl]-1-ethyl-3,3-dimethyl

[0055] Purified 2-(2'-hydroxyphenyl)-4-chloromethyloxazole (0.63 g, 3.0 mmol) was added to an anhydrous CH3CN (25 mL) solution of 4-(diethylamino)-2-hydroxybenzaldehyde (0.58 g, 3.0 mmol) and anhydrous potassium carbonate (0.83 g, 6.0 mmol). Potassium iodide (0.005 g, 0.03 mmol) was then added to the above solution. The reaction mixture was refluxed under N2 protection for 10 hours. The solution was then cooled to room temperature and most of the solvent was removed under reduced pressure. The residue was dissolved in CHCl3 (200 mL) and washed with brine (200 mL). The organic phases were combined and dried over anhydrous Na2SO4. Finally, the solvent was evaporated to dryness, and the residue was purified by column chromatography (ethyl acetate / petroleum ether (v / v, 1:6)) to give the pure product (0.61 g, yield: 55%).

[0056] 4) Synthesis: Fluorescent molecule H-HPMO

[0057] The synthesis steps of H-HPMO are as follows: The purified compound (0.50 g, 1.4 mmol) and hemicyanin (0.49 g, 1.4 mmol) from Example 3 were dissolved in 20 mL of CH3OH, and the mixture was stirred at 65 °C for 9 hours, then cooled to room temperature. After removing most of the solvent by vacuum evaporation, the reaction mixture was recrystallized at –20 °C. The resulting green precipitate was washed three times with 50 mL of ice-cold ethanol each time. The pure product was obtained as green crystals (0.71 g, yield: 46%), which were dried and stored in the dark. The structure was confirmed as the expected product by characterization by 1H NMR, 1C NMR, high-resolution mass spectrometry, and infrared spectroscopy. Figure 2 , 3, 4, 5.

[0058] Example 2. Detection of tyrosine phosphorylated peptides in solution

[0059] Accurately weigh phosphorylated peptides 1pY, 2pY, and 3pY containing different numbers of tyrosine residues, and dissolve non-phosphorylated peptide NMP in 1 mL of ultrapure water to prepare 5 mmol·L⁻¹ NMP solutions. -1 The stock solution was then prepared separately at 0.01 mol·L⁻¹. -1 Cu(NO3)2 aqueous solution and 0.01 mol·L -1 The H-HPMO solution (dimethyl sulfoxide (DMSO) was used as the solvent). First, 10 μL of the H-HPMO solution was added to 3 mL of HEPES (50 mmol·L⁻¹). -1Add 10 μL of Cu(NO3)2 aqueous solution to the buffer solution, mix thoroughly, and record the initial fluorescence emission spectrum using a fluorescence spectrometer. Then, add 60 μL of tyrosine phosphorylated peptide 1pY, 2pY, 3pY and non-phosphorylated peptide NMP stock solutions respectively, and record the fluorescence emission spectra. Maintain consistent parameters during testing, with the excitation wavelength set to 500 nm.

[0060] like Figure 6 As shown, the addition of tyrosine-phosphorylated peptides 1pY, 2pY, and 3pY all induced significant fluorescence recovery, while the addition of the non-phosphorylated peptide NMP did not result in any fluorescence change, indicating that the probe H-HPMO-Cu... 2+ It can be used to detect tyrosine-phosphorylated peptides, but has no response to non-phosphorylated peptides.

[0061] Example 3. Quantitative detection of tyrosine phosphorylated peptides

[0062] First, prepare the H-HPMO standard solution (0.01 mol·L⁻¹). -1 Take 10 μL and add it to 3 mL of HEPES (50 mmol·L⁻¹). -1 Add 10 μL of Cu(NO3)2 aqueous solution (0.01 mol·L⁻¹) to the buffer solution. -1 After thorough mixing, the initial fluorescence value A at 598 nm was recorded using a fluorescence spectrometer. Then, eight standard aqueous solutions of tyrosine phosphorylated peptide 1pY with different concentrations (33 μmol·L⁻¹) were prepared. –1 37.5 μmol·L –1 45 μmol·L –1 54 μmol·L –1 58 μmol·L –1 62 μmol·L –1 66 μmol·L –1 ), and 60 μL of the above solution was added to a solution containing the probe H-HPMO–Cu. 2+ (33 μmol·L) -1 The tyrosine phosphorylated peptide was mixed thoroughly in HEPES buffer solution and the fluorescence value (B) at 598 nm was recorded using a fluorescence spectrometer. A standard curve was plotted based on the concentration of the tyrosine phosphorylated peptide and the corresponding fluorescence ratio (B / A).

[0063] like Figure 7 As shown, standard curves were plotted by comparing different concentrations of tyrosine phosphorylated peptides with their corresponding fluorescence ratios. Unknown concentrations of tyrosine phosphorylated peptides were added to a solution containing H-HPMO–Cu. 2+ (33 μmol·L) -1In the test solution of ), under the same conditions, the fluorescence intensity value B1 at 598 nm was recorded. Substituting B1 / A into the standard curve, its concentration can be calculated.

[0064] Example 4. Determination of detection limit

[0065] H-HPMO standard solution (0.01 mol·L⁻¹) -1 Take 10 μL and add it to 3 mL of HEPES (50 mmol·L⁻¹). -1 Add 10 μL of Cu(NO3)2 aqueous solution (0.01 mol·L⁻¹) to the buffer solution. -1 Record the initial fluorescence spectrum, and then add 2 μL (0.005 mol·L⁻¹) to the previous solution each time. -1 Tyrosine-phosphorylated peptides were recorded sequentially, with the fluorescence emission spectra recorded 20 times. Figure 8 As shown in the figure. A graph was plotted between the ratio of fluorescence intensity at 598 nm and the corresponding concentration of tyrosine-phosphorylated peptide, as shown in the figure. Figure 9 As shown, the detection limit is calculated using the formula (detection limit = 3 standard deviations / slope) to be 1.10 × 10⁻⁶. -7 mol·L -1 .

[0066] Example 5. Effects of common ions on the fluorescence properties of the probe H-HPMO-Cu2+

[0067] Prepare 0.1 mol·L⁻¹ solutions respectively -1 The solution contains magnesium chloride, calcium chloride, sodium chloride, sodium carbonate, sodium sulfate, sodium nitrate, potassium isothiocyanate, disodium adenosine triphosphate, and disodium adenosine diphosphate. 10 μL of each of these solutions is added to 3 mL of HEPES (50 mmol·L⁻¹). -1 Add 10 μL of H-HPMO and Cu(NO3)2 standard solutions (0.01 mol·L⁻¹) to the buffer solution, respectively. -1 After thorough mixing, the fluorescence emission spectrum was recorded using a fluorescence spectrometer. All parameters were kept consistent during testing, with the excitation wavelength set to 500 nm.

[0068] like Figure 10 As shown, after adding the above ions, the probe H-HPMO–Cu 2+ The fluorescence emission spectra of the probe H-HPMO–Cu showed no significant changes, indicating that other common ions do not affect the probe. 2+ This affects the fluorescence properties.

[0069] Example 6. Real-time monitoring of protein kinase activity

[0070] Accurately weigh 7 mg of peptide substrate EE-13 (EAIYAAPAAYIAE) and dissolve it in 1 mL of ultrapure water to prepare 5 mmol·L⁻¹ -1 The stock solution was prepared the night before the assay and stored in a biological freezer at -8°C. Then, 0.01 mol·L⁻¹ solutions were prepared separately. -1 Cu(NO3)2 stock solution, 0.1 mol·L -1 MgCl2 stock solution, 0.1 mol·L -1 NaCl stock solution and 0.1 mol·L -1 Adenine nucleoside triphosphate disodium (ATP) 2- The standard stock solution is stored at -20°C. Prepare a 25 mmol / L solution. –1 Tris–HCl buffer solution and HCl solution (0.1 mol·L⁻¹) –1 Adjust it to pH 7.4.

[0071] Before testing, first take 10 sterile 2mL centrifuge tubes, then take 2255μL of freshly prepared Tris-HCl (25mmol·L⁻¹). –1 Add buffer to a 2 mL centrifuge tube, and add 120 μL of peptide substrate EE-13 standard stock solution (5 mmol·L⁻¹). -1 Add 15 μL of adenine nucleoside triphosphate disodium (ATP) to it, and then add 15 μL of adenine nucleoside triphosphate disodium (ATP). 2- 0.1 mol·L -1 ), 300 μL MgCl2 (0.1 mol·L -1 ) and 300 μL NaCl (0.1 mol·L -1 Add to the above substrate solution. Add 10 μL of c-Abl kinase stock solution (0.24 μg / μL) to a final concentration of 100 nmol·L⁻¹. –1 At this point, the kinase reaction begins. After incubating the above reaction mixture in a constant temperature metal bath at 37°C for 5 min, 200 μL of the reaction solution was added to each of the black 96-well plates. Simultaneously, 1.5 μL (0.001 mol·L⁻¹) was rapidly added. -1 H-HPMO and Cu 2+ A mixed solution (composition: H-HPMO, copper nitrate, and H2O) was prepared, and the fluorescence intensity value B was recorded using a microplate reader (the microplate reader parameters were pre-set: temperature controlled at 37℃, dual-track vibration mode (medium speed), data read from the bottom, excitation wavelength: 560nm, emission wavelength: 598nm, test mode: endpoint kinetics). Fluorescence readings were taken every 5 minutes for 80 minutes, and each experiment was performed 3 times to obtain the final results.

[0072] In another blank control experiment, 10 μL of c-Abl kinase stock solution was replaced with 10 μL of H2O. The contents of other additives were the same as those in the above experimental group. The same steps were used to record the change of fluorescence intensity over time under the same conditions using an ELISA reader. The fluorescence value was recorded as the initial fluorescence value A.

[0073] like Figure 11 After adding c-Abl kinase, the fluorescence value B of the reaction mixture was 12756 at 60 min, while the fluorescence value A of the reaction mixture without kinase was 3254 at 60 min. The B / A ratio was 3.92, indicating that the probe H-HPMO–Cu 2+ It can be used to monitor protein kinase activity.

[0074] Example 7. Screening of protein kinase inhibitors

[0075] Add 2255 μL of freshly prepared Tris-HCl (25 mmol·L⁻¹) to a 2 mL centrifuge tube. –1 Add 120 μL of peptide substrate EE-13 standard stock solution (prepared in Example 6) and 15 μL of adenine nucleoside triphosphate disodium (ATP) buffer (pH=7.4), then add 120 μL of EE-13 standard stock solution (prepared in Example 6) and 15 μL of adenine nucleoside triphosphate disodium (ATP) buffer. 2- 0.1 mol·L -1 ), 300 μL MgCl2 (0.1 mol·L -1 ) and 300 μL NaCl (0.1 mol·L -1 After mixing thoroughly, 200 μL was added to a black 96-well plate, and the initial fluorescence value A at 598 nm was recorded after 60 min.

[0076] Add the same amount of substrate peptide and various salt solutions (with the same composition as above) to another 2 mL centrifuge tube, then add 10 μL of c-Abl (0.24 μg / μL) kinase stock solution to a final concentration of 100 nmol·L⁻¹. –1 At this point, 10 μL of the candidate kinase inhibitor is quickly added. After incubating the above reaction mixture in a constant temperature metal bath at 37°C for 5 min, 200 μL of the reaction solution is added to each of the black 96-well plates. H-HPMO and Cu are then quickly added simultaneously. 2+ (33 μmol·L) -1 A mixed solution (composition: H-HPMO, copper nitrate and H2O) was prepared, and the fluorescence intensity value B1 at 598nm was recorded at 60min using an ELISA reader (the ELISA reader parameters were set in advance, the temperature was controlled at 37℃, the dual-track mode vibration (medium speed) was used, the data was read from the bottom, the excitation wavelength was 560nm, and the test mode was endpoint kinetics).

[0077] like Figure 12The fluorescence values ​​at 60 min after adding candidate kinase inhibitors such as huperzine B, allicin, costunolide, dehydrocostunolide, dehydroevodiamine hydrochloride, imatinib, delphinidin, and nordecibelone were 4.044517725, 3.720939819, 5.049464138, 3.809150866, 4.029266282, 1.398186315, 3.850370981, and 5.234542457, respectively. Among them, 1.398186315 is much smaller than 0.5B / A in Example 6, indicating that it has an inhibitory effect, corresponding to the candidate inhibitor imatinib.

[0078] Matters not covered in this invention are common knowledge.

[0079] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of dye molecules as fluorescent probes in the detection of tyrosine phosphorylated peptides; The dye molecule was named H-HPMO–Cu. 2+ Its structural formula is as follows: .

2. The application according to claim 1, characterized in that, Specifically, the following steps are included: 1) Using excitation light with wavelengths from 450 to 560 nm, the presence of H-HPMO–Cu was detected by fluorescence spectroscopy. 2+ The initial fluorescence value A of the solution at any point between 590 and 600 nm; The H-HPMO–Cu 2+ The composition of the solution is: 3 to 300 μmol·L⁻¹ –1 The dye H-HPMO, 3 to 300 μmol·L –1 Copper nitrate, hydroxyethylpiperazine ethanethiosulfonic acid (HEPES), concentration 10 to 50 mmol·L⁻¹ –1 pH = 6.8 to 7.4; 2) Add the substrate peptide to be detected to a solution containing H-HPMO–Cu 2+ In the solution; using excitation light with a wavelength of 450 to 560 nm, the fluorescence value B of the solution containing the substrate peptide at the same point as in step 1) was detected by fluorescence spectroscopy; the substrate peptide solution and the probe H-HPMO–Cu 2+ The molar ratio of the solutions is between 2:1 and 5:1; If B is greater than or equal to 3A, it means the substrate peptide is a phosphorylated peptide; if B is equal to A or less than A, it means the substrate peptide is a non-phosphorylated peptide.

3. The application according to claim 1, characterized in that, 1) Using excitation light with wavelengths from 450 to 560 nm, the presence of H-HPMO–Cu was detected by fluorescence spectroscopy. 2+ The initial fluorescence value A of the solution at any point between 590 and 600 nm; The H-HPMO–Cu 2+ The composition of the solution is: 3 to 300 μmol·L⁻¹ –1 The dye H-HPMO, 3 to 300 μmol·L –1 Copper nitrate, hydroxyethylpiperazine ethanethiosulfonic acid (HEPES), concentration 10 to 50 mmol·L⁻¹ –1 pH = 6.8 to 7.4; 2) Different concentrations from 10 to 80 μmol·L –1 Tyrosine phosphorylated peptides are added to H-HPMO–Cu 2+ In the solution, at least five test solutions containing different concentrations of tyrosine phosphorylated peptides were obtained; using excitation light with a wavelength of 450 to 560 nm, the fluorescence value B of the test solutions at the same point as in step 1) at 590 to 600 nm was detected by a fluorescence spectrometer; a standard curve was plotted with the concentration of tyrosine phosphorylated peptides and B / A as the x and y axes, respectively. 3) Add the peptide sample to be tested to a solution containing H-HPMO–Cu 2+ The solution containing phosphorylated peptides was used as the test solution; the fluorescence value B1 of the solution containing phosphorylated peptides was detected by a fluorescence spectrometer under the same conditions as in step 2) using excitation light with a wavelength of 450 to 560 nm. Substituting the fluorescence value B1 / A into the above standard curve yields the concentration of the tyrosine phosphorylated peptide to be tested in the test solution.

4. The application of dye molecules as fluorescent probes in monitoring protein kinase activity, characterized in that, Specifically, the following steps are included: 1) Add the substrate peptide solution to a solution containing H-HPMO–Cu 2+ In a solution containing the substrate peptide, H-HPMO–Cu was detected using an enzyme-linked immunosorbent assay (ELISA) reader with excitation light at a wavelength of 450 to 560 nm. 2+ The initial fluorescence value A of the solution at any point between 590 and 600 nm; The H-HPMO–Cu 2+ The solution composition is 10 to 25 mmol·L⁻¹ –1 Tris–HCl, 0.05 to 0.30 mmol·L –1 EE-13 substrate peptide, 0.1 to 0.5 mmol·L⁻¹ –1 Adenine nucleoside triphosphate, 1 to 10 mmol·L –1 MgCl2, 1 to 10 mmol·L –1 NaCl, 20 to 60 μmol·L –1 H-HPMO–Cu 2+ ; Substrate peptide EE-13 solution and H-HPMO–Cu 2+ The molar ratio of the solutions is between 2:1 and 5:1; 2) Add the protein kinase to be tested to the solution containing the substrate peptide in step 1), use excitation light with a wavelength of 450 to 560 nm, and use the same conditions as in step 1) to detect the fluorescence value B of the solution containing the protein kinase between 0 and 60 min using an ELISA reader, and read the results every 5 minutes. The concentration of the protein kinase to be tested in the added protein kinase solution was 50 to 100 nmol·L⁻¹. –1 ; If B is greater than or equal to 2A after 60 minutes, it indicates that the probe H-HPMO–Cu 2+ It can detect the process by which protein kinase catalyzes the formation of tyrosine phosphorylated peptides from the substrate peptide EE-13; if B is less than 2A, it means that this process cannot be monitored. The substrate peptide EE-13 has the following structural formula: The dye molecule is named H-HPMO–Cu. 2+ Its structural formula is as follows: .

5. The application of a dye molecule as a fluorescent probe in screening protein kinase inhibitors, characterized in that, 1) Add the substrate peptide EE-13 to a solution containing H-HPMO–Cu 2+ In the solution; using excitation light with a wavelength of 450 to 560 nm, the presence of H-HPMO–Cu was detected by an enzyme-linked immunosorbent assay (ELISA) reader. 2+ The fluorescence intensity at any point between 590 and 600 nm in the solution between 50 and 60 min is taken as the initial fluorescence value A; The H-HPMO–Cu 2+ The solution composition is 10 to 25 mmol·L⁻¹ –1 Tris–HCl, 0.05 to 0.30 mmol·L –1 EE-13 substrate peptide, 0.1 to 0.5 mmol·L⁻¹ –1 Adenine nucleoside triphosphate, 1 to 10 mmol·L –1 MgCl2, 1 to 10 mmol·L –1 NaCl, 20 to 60 μmol·L –1 H-HPMO–Cu 2+ ; Substrate peptide EE-13 solution and H-HPMO–Cu 2+ The molar ratio of the solutions is between 2:1 and 5:1; 2) Add a protein kinase that can catalyze the conversion of the substrate peptide to tyrosine phosphorylated peptide to the solution containing the substrate peptide in step 1). Use excitation light with a wavelength of 450 to 560 nm and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the fluorescence value B at the same point as in step 1) at 590 to 600 nm between 50 and 60 min. The concentration of the protein kinase to be tested in the added protein kinase solution was 50 to 100 nmol·L⁻¹. –1 ; 3) Add the protein kinase that can catalyze the conversion of substrate peptide EE-13 to tyrosine phosphorylated peptide in step 2) and the kinase inhibitor to be screened to the solution containing substrate peptide EE-13 in step 1). Use excitation light with a wavelength of 450 to 560 nm and detect the fluorescence value B1 of the solution under the same conditions as in step 2) between 50 and 60 min using an enzyme-linked immunosorbent assay (ELISA) reader. The concentration of the protein kinase to be tested in the added protein kinase solution was 50 to 100 nmol·L⁻¹. –1 ; The concentration of candidate kinase inhibitors added to the solution of the kinase inhibitor to be screened is 5 to 10 μmol·L⁻¹ –1 ; If B1 / A is greater than or equal to 0.5 B / A, it means that the kinase inhibitor to be screened has no inhibitory effect; If B1 / A is less than 0.5 B / A, it means that the kinase inhibitor to be screened has an inhibitory effect; The dye molecule was named H-HPMO–Cu. 2+ Its structural formula is as follows: .

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