Fluorescent Probe Targeting SHP2, Preparation Method Thereof and Application Thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a fluorescent probe targeting SHP2, a preparation method, and its application. The present invention determines the core pharmacophore by replacing the amino group in the central region B of SHP2-099 with a para-methyl group and introducing a sulfur atom as a connecting atom, and integrates the SBD fluorophore therein to develop a series of fluorescent probes SHP-PS (1-4) targeting SHP2 with connecting arms of different lengths. The series of fluorescent probes targeting SHP2 provided by the present invention have high specificity and good target affinity, can selectively label SHP2 proteins in living cells, and have an environmentally sensitive switching mechanism, providing new tools for studying the role of SHP2 in signal transduction and its significance in disease progression, and are expected to accelerate the research of diseases such as cancer and provide new approaches for diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent probe targeting SHP2, a preparation method and an application thereof. Background Art

[0002] SHP2 is a non-receptor protein tyrosine phosphatase belonging to the PTP family. It plays a crucial role in cell signal transduction, participating in cell survival, proliferation, and migration, and regulating a range of cellular signals, including metabolism, growth, differentiation, transcription, and cancerous transformation. Furthermore, SHP2 plays a central role in oncogenic signaling pathways that include, but are not limited to, interactions with immune checkpoints such as JAK / STAT, PI3K / AKT, RAS / Raf / MAPK, and PD-1 / PD-L1. It is crucial for mediating biological responses to growth factors, hormones, cytokines, and cell adhesion molecules, as well as signal transduction pathways that control developmental processes and hematopoiesis.

[0003] Dysregulation of SHP2, manifested by overexpression or hyperactivation, has been associated with a range of diseases and is frequently detected in various solid tumors, making it a promising target for cancer therapy.

[0004] Existing SHP2 detection methods, such as immunoblotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and substrate-based assays, have greatly enhanced our understanding of SHP2 biology. However, these methods are often limited by low throughput, low sensitivity, and the inability to monitor SHP2 in real time within living cells.

[0005] In recent years, fluorescence imaging has become increasingly important in the field of target visualization. Small molecule fluorescent probes have emerged as a powerful analytical tool and are now widely used in the labeling, observation, and detection of proteins, ions, nucleic acids, and the physical microenvironment of cells.

[0006] However, to date, no small molecular fluorescent probe specific for SHP2 has been reported. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a fluorescent probe targeting SHP2, a preparation method and an application thereof.

[0008] The novel small molecule fluorescent probe provided by the present invention can track SHP2 in real time using imaging technology with high sensitivity and spatiotemporal resolution. It has an environmentally sensitive switching mechanism, high specificity, good binding affinity, and can selectively label SHP2 in living cells.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention is to provide a fluorescent probe targeting SHP2, wherein the fluorescent probe has the following structure:

[0011]

[0012] Wherein, n=1-10.

[0013] The second aspect of the present invention is to provide a method for preparing the fluorescent probe targeting SHP2, which specifically comprises the following steps:

[0014] (1) Reaction between pharmacophore and fluorophore

[0015] The pharmacophore, fluorophore, and N,N-diisopropylacetic acid are dissolved in acetonitrile and reacted at room temperature to obtain a fluorescent probe, wherein the molar ratio of pharmacophore:fluorophore:N,N-diisopropylacetic acid is 1:1-2:1-1.5. The reaction equation is shown below;

[0016]

[0017] (2) trifluoroacetic acid is used to remove the protecting group of the fluorescent probe prepared in (1) to prepare a fluorescent probe targeting SHP2, as shown in the following equation;

[0018]

[0019] In the above formula, R1 is independently selected from H, Cl, Br or I;

[0020] R2 is independently selected from H or tert-butyloxycarbonyl; n=1-10.

[0021] Preferably, n=2, 5, 7, 9.

[0022] In the above-mentioned method for preparing the fluorescent probe targeting SHP2, the method for preparing the pharmacophore is:

[0023] (I) 2-chloro-3-fluoroaniline and tert-butyl mercaptan are reacted under alkaline conditions to prepare compound 2;

[0024] (II) heating the compound 2 obtained in (I) in concentrated hydrochloric acid to remove the tert-butyl group to obtain compound 3;

[0025] (III) cross-linking 2-bromo-5-chloro-3-methylpyrazine with the compound 3 obtained in (II) to obtain compound 4;

[0026] (IV) reacting tert-butyl carbamate (4-methylpiperidin-4-yl) with compound 4 obtained in (III) to obtain compound 5;

[0027] (V) Compound 6, i.e., the pharmacophore, is obtained by reacting compound 5 obtained in (IV) with bromoacetyl chloride.

[0028] Preferably, in the preparation method of the pharmacophore, the reaction conditions of each step are as follows:

[0029] (I) 2-chloro-3-fluoroaniline and tert-butyl mercaptan are reacted in a molar ratio of 1:2 to 4 in the presence of Cs2CO3 and in N,N-dimethylformamide as solvent at 100 to 150°C;

[0030] The heating temperature in (II) is 60 to 100° C.;

[0031] (III) a molar ratio of 2-bromo-5-chloro-3-methylpyrazine to compound 3 of 1:1-2, and a cross-linking reaction is carried out in the presence of CuI, 1,10-phenanthroline, K3PO4, and dioxane at a temperature of 70-150°C;

[0032] (IV) reacting tert-butyl (4-methylpiperidin-4-yl)carbamate, N,N-diisopropylethylamine, and compound 4 obtained in (III) at 80-120° C. in the presence of dimethyl sulfoxide, wherein the molar ratio of compound 4: tert-butyl (4-methylpiperidin-4-yl)carbamate: N,N-diisopropylethylamine is 1.8-3:1:2-4;

[0033] (V) The molar ratio of compound 5 to bromoacetyl chloride is 1:5-7.

[0034] Preferably, the preparation method of the fluorophore is as follows:

[0035] Using 4-chlorobenzofuran as the raw material, compounds 8 and 9 were obtained in sequence through a two-step substitution reaction with chlorosulfonic acid and dimethylamine. The obtained compound 9 was then connected with a fatty diamine to obtain compound 10, the fluorophore.

[0036] Further preferably, the preparation conditions of the fluorophore are as follows:

[0037] The reaction temperature of 4-chlorobenzofuran and chlorosulfonic acid is 100-150°C;

[0038] The molar ratio of compound 9 to the fatty diamine is 1:15-20.

[0039] The third aspect of the present invention provides the use of a series of fluorescent probes targeting SHP2 in SHP2 detection.

[0040] The beneficial effects of the present invention are:

[0041] (1) Provided are a series of small molecule fluorescent probes SHP-PS (1-4) targeting the SHP2 protein. These fluorescent probes exhibit excellent optical properties and biocompatibility, high signal-to-noise ratio, and stable fluorescence signals, which can meet the requirements of imaging. Moreover, these fluorescent probes maintain good affinity for SHP2 and can specifically label SHP2 in living cells, showing excellent protein selectivity. Homologous proteins such as SHP1 and other proteins have minimal interference with imaging.

[0042] In addition, these fluorescent probes have selective switching functions, are easy to use, and do not require washing before imaging. Cell imaging shows that they can clearly locate the SHP2 protein;

[0043] (2) A method for preparing such probes has been developed, which can achieve large-scale synthesis of such probes, which will help enhance researchers' understanding of the role of SHP2 in signal transduction and its significance in disease progression, thereby accelerating research and potentially revealing new diagnostic and therapeutic pathways for conditions such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a design diagram of the fluorescent probe targeting SHP2 in Example 1 of the present invention;

[0045] Figure 2 This is a diagram of the synthesis method of a series of fluorescent probes SHP-PS (1-4) targeting SHP2 in Example 2 of the present invention;

[0046] Figure 3 The emission spectra (A1, A2, A3, A4) of the series of fluorescent probes SHP-PS (1-4) targeting SHP2 in Example 3 of the present invention in solvents of different polarity (water, methanol, ethanol, propanol, butanol) and in systems of different viscosities (B1, B2, B3, B4);

[0047] Figure 4 : This is the in vitro inhibition curve of different concentration series of fluorescent probes SHP-PS (1-4) and SHP-099 on SHP2 in Example 4 of the present invention;

[0048] Figure 5 Bright-field imaging (L) and fluorescence imaging (R) of SHP-PS1 (A), SHP-PS2 (B), SHP-PS3 (C), and SHP-PS4 (D) in Example 5 of the present invention, wherein A1-D1 represent the fluorescent probe (2 μmol / L), and A2-D2 represent the fluorescent probe (2 μmol / L) + SHP-099 (20 μmol / L); the scale bar is 50 μm;

[0049] Figure 6The flow cytometry method for detecting the binding of the fluorescent probe to SHP2 in living cells in Example 6 of the present invention is as follows;

[0050] Figure 7 The fluorescence turn-on characteristics of the fluorescent probe SHP-PS2 after incubation of SHP2 protein or living cells at different concentrations at 37° C. for 30 minutes in Example 7 of the present invention;

[0051] Figure 8 This is a graph showing the fluorescence intensity (1 μmol / L) of different proteins and fluorescent probes after incubation for 30 min at 37° C. in Example 8 of the present invention;

[0052] Figure 9 The fluorescence polarization values of the fluorescent probe and SHP2 protein (B) at different concentrations detected by the fluorescence polarization method in Example 9 of the present invention are shown. DETAILED DESCRIPTION

[0053] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0054] It should be noted that the terms used in the embodiments of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not specified in the following specific embodiments, conventional methods and conditions within the art are generally followed.

[0055] In a typical embodiment of the present invention, the design of the fluorescent probe includes a structural optimization process of SHP-099, wherein the activity is enhanced by replacing the amino group in the central region B of SHP-099 with a p-methyl group (RWC-4550) and introducing a sulfur atom as a linker atom (TNO-155). Finally, structure 1 is determined as the pharmacophore, and the SBD fluorophore is introduced therein. A series of fluorescent probes SHP-PS with different linker arm lengths are designed and synthesized.

[0056] In another typical embodiment of the present invention, a series of fluorescent probes are chemically synthesized, involving synthesizing a pharmacophore and a fluorophore from the starting materials through a series of substitution and cross-linking reactions, connecting the two through a nucleophilic reaction, and finally deprotecting to obtain the target fluorescent probe.

[0057] In addition, the present invention also measured the optical properties of a series of fluorescent probes, such as ultraviolet absorption, fluorescence excitation and emission wavelengths. In addition, the environmental sensitivity of the fluorescent probes was also evaluated.

[0058] Furthermore, the present invention also evaluated the inhibitory ability of the fluorescent probe on the enzyme activities of SHP1 and SHP2. By adding the substrate DiFMUP after pre-incubation with the enzyme and measuring the fluorescence signal, the results showed that the fluorescent probe had a strong inhibitory effect on SHP2 but had no effect on SHP1, demonstrating the selective labeling ability for SHP2. Although the introduction of the fluorophore may affect the inhibitory activity, the fluorescent probe still exhibited strong binding ability and satisfactory selectivity for SHP2.

[0059] Another embodiment of the present invention describes a fluorescent probe for specifically labeling SHP2 in living cells. Direct imaging after incubation revealed that all fluorescent probes specifically bound to SHP2. SHP-PS2 performed best in cell imaging, exhibiting both good SHP2 inhibitory activity and environmental sensitivity. While SHP-PS1 exhibited the best inhibitory activity, its imaging results were the worst, presumably due to its short linker, which prevented the fluorophore from entering the hydrophobic environment of the binding site. Furthermore, flow cytometry experiments validated the fluorescent probe's ability to specifically label SHP2 in living cells. Results showed a significant increase in cell fluorescence intensity after probe incubation, and a decrease in fluorescence intensity under competition with SHP-099, confirming the specific binding of the fluorescent probe to SHP2.

[0060] In addition, the present invention also verified the switching properties of the fluorescent probe, which was activated and released strong fluorescence by binding to the SHP2 protein and living cells. It was found that the performance of the fluorescent probe was affected by the length of the connecting arm, among which SHP-PS1 performed poorly, while SHP-PS2, SHP-PS3 and SHP-PS4 showed good switching properties.

[0061] In addition, the selectivity of the fluorescent probe for SHP2 protein was further verified through experiments. The results showed that compared with other proteins, the binding of the fluorescent probe to SHP2 led to a significant increase in fluorescence intensity, and the fluorescence intensity decreased under competition with SHP-099, confirming the high selectivity and specific binding of the obtained fluorescent probe to SHP2.

[0062] Furthermore, the present invention utilizes fluorescence polarization technology to accurately reflect the interaction between molecules by measuring the change in fluorescence polarization value after the fluorescent probe binds to the SHP2 protein, which is used for affinity determination and the establishment of competitive fluorescence screening methods. The results show that the prepared fluorescent probe has a strong affinity for SHP2, which is consistent with the results of SHP2 enzyme activity inhibition.

[0063] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0064] Example 1 Design of fluorescent probe

[0065] The design route of the fluorescent probe provided by the present invention is shown in the attached Figure 1 As shown, attached Figure 1 In the figure, Structure 1 represents the structure 1, Fharmacophore represents the pharmacophore, Fluorophore represents the fluorophore, SHP-PS represents the series of fluorescent probes, Formula 1 represents the two-dimensional structure of SHP-099, and in Formula 1, A represents the hydrophobic region, B represents the central region, C represents the polar region, and L represents the connecting atom.

[0066] From a two-dimensional structural perspective, SHP-099 is mainly divided into a hydrophobic region A, a central region B, and a polar region C. If the synthesized fluorescent probe uses a similar structure to SHP-099 as a pharmacophore, the binding pocket where the pharmacophore binds to the protein will be flanked by large-surface solvent-soluble regions, providing potential binding sites for the optimization of the inhibitor's molecular structure.

[0067] RWC-4550 is a compound obtained by replacing the 2-amino group of the pyrazine ring in the middle B region of SHP099 with a p-methyl group, which improves the activity.

[0068] TNO-155 introduces sulfur as a linker atom, increasing the flexibility and activity of the molecule.

[0069] Taking into account the pharmacophore activity and synthetic feasibility, the present invention determined that structure 1 was the pharmacophore, introduced the SBD fluorophore into its hydrophobic region, and designed and synthesized a series of fluorescent probes SHP-PS with linkers of different lengths.

[0070] Example 2 Chemical synthesis of probe

[0071] A typical fluorescent probe consists of a fluorophore, a pharmacophore and a linker between the two. The chemical synthesis method of the probe is as follows. Figure 2 The specific steps are as follows:

[0072] (1) Reaction between pharmacophore and fluorophore

[0073] Chemical synthesis of S1 pharmacophore

[0074] (I) 68.7 mmol of 2-chloro-3-fluoroaniline, 206 mmol of 2-methyl-2-propanethiol, and 206 mmol of cesium carbonate were added to 100 mL of N,N-dimethylformamide (DMF), and the mixture was stirred at 120° C. for 24 h to obtain compound 2 as a yellow oil (yield 85.7%).

[0075] (II) Compound 2 (55.6 mmol) obtained in (I) was added to hydrochloric acid (100 mL) and stirred at 80° C. for 6 h to obtain compound 3 as a white solid (yield 92.3%);

[0076] (III) 2.50 g of compound 3, 2.60 g of 2-bromo-5-chloro-3-methylpyrazine, 597 mg of CuI, 1.13 g of o-phenanthroline, and 9.97 g of K3PO4 were added to 100 mL of 1,4-dioxane, and then stirred at 90°C under nitrogen for 10 h to obtain compound 4 (yield 45.3%).

[0077] (IV) 1.50 g of tert-butyl (4-methylpiperidin-4-yl)carbamate and 1.87 mL of diisopropylethylamine (DIPEA) were added to a solution of 1.00 g of compound 4 in 15 mL of dimethyl sulfoxide (DMSO), and the mixture was stirred at 100 °C for 3 h to obtain compound 5 (yield 77.2%).

[0078] (V) A solution of 1.00 g of compound 5 and 2.14 mL of DIPEA in 20 mL of dichloromethane (DCM) was cooled in an ice bath, and then 1.08 mL of bromoacetyl chloride was added dropwise. The mixture was stirred at room temperature for 3 h, and then n-hexane was added. The precipitate was filtered to obtain the pharmacophore compound 6 as a yellow solid (yield 88.9%).

[0079] Preparation of S2 fluorophore

[0080] 3.00 g of 4-chlorobenzofuran was slowly added portionwise to 30 mL of chlorosulfonic acid under an ice bath, and then the mixture was transferred to 120° C. and reacted for 6 h to obtain compound 8 as an off-white solid (yield 93.5%);

[0081] 4.00 g of compound 8 was dissolved in 40 mL of acetonitrile, and then a mixture of dimethylamine hydrochloride, triethylamine, and acetonitrile was added. The reaction mixture was then stirred in an ice bath for 30 min and then at room temperature for 3 h to obtain compound 9 as a white solid (yield 73.7%).

[0082] 1.00 g of compound 9 was dissolved in 50 mL of acetonitrile with fatty diamines of different lengths (1,3-propylenediamine, 1,6-hexanediamine, 1,8-octanediamine, 1,10-decanediamine) and stirred at 60 °C for 6 h to obtain fluorophore compounds 10 with linkers of different lengths.

[0083] 200 mg of compound 6, 153 mg of compound 10 and 133 mg of DIPEA were dissolved in 50 mL of acetonitrile and stirred at room temperature for 20 h to obtain the fluorescent probe compound 11.

[0084] (2) Using trifluoroacetic acid to remove the protecting group of the fluorescent probe compound 11 prepared in (1), a fluorescent probe targeting SHP2 was prepared. The specific operation was as follows:

[0085] Trifluoroacetic acid (TFA) was added dropwise to a DCM solution of compound 11, and the mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM and methanol, then washed with saturated sodium bicarbonate solution. The crude product was purified by silica gel column chromatography to obtain a series of fluorescent probes targeting SHP2, SHP-PS (1-4). The yields of SHP-PS (1-4) were 73.9%, 84.5%, 54.2%, and 66.4%, respectively, with increasing linker length.

[0086] Example 3 Measurement of optical properties of probes

[0087] The optical properties of the fluorescent probe, such as ultraviolet absorption, fluorescence excitation and emission wavelength, were measured, as shown in Table 1. In Table 1, Φ is the relative quantum yield calculated with sodium fluorescein as a reference, λ max is the maximum UV absorption wavelength, λ ex is the maximum fluorescence excitation wavelength, λ em is the wavelength of maximum fluorescence emission.

[0088] Table 1 Optical properties of fluorescent probes

[0089]

[0090] As shown in Table 1, various fluorescent probes exhibit two distinct UV absorption bands at 355 nm and 445 nm. The band at 355 nm corresponds to the conjugated system of the pharmacophore, which is known for its minimal fluorescence emission and is not the focus of the present invention.

[0091] Additionally, all fluorescent probes exhibited maximum excitation at 440 nm and 445 nm and peaked at 570 nm, resulting in a Stokes shift of approximately 130 nm, which is advantageous because it can reduce background interference, minimize phototoxicity to biological samples, and improve detection sensitivity.

[0092] In addition, the present invention also evaluates the environmental sensitivity of various fluorescent probes. Figure 3 Emission spectra (A1, A2, A3, A4) of each fluorescent probe SHP-PS (1-4) in solvents of different polarity (water, methanol, ethanol, propanol, butanol) and in systems with different viscosities (B1, B2, B3, B4).

[0093] Figure 3In the figure, the ordinate is the emission / absorption intensity, and the abscissa is the wavelength. The viscosity system is realized by a mixture of glycerol and water, where the percentage represents the proportion of the glycerol volume to the total volume.

[0094] Figure 3 The results show a positive correlation between the fluorescence intensity of each fluorescent probe and decreasing solvent polarity and increasing viscosity. Furthermore, the quantum yield of the probe is significantly higher in low-polarity environments (see Table 1 for details). Notably, the length of the linker between the pharmacophore and the fluorophore significantly influences the environmental sensitivity of the fluorescent probe. For example, the probes SHP-PS1 and SHP-PS2 exhibit excellent sensitivity, which decreases with increasing linker length, demonstrating the importance of optimizing the linker length in probe design.

[0095] Example 4 Enzyme Activity Inhibition Test of Fluorescent Probe

[0096] The ability of fluorescent probes to inhibit SHP1 and SHP2 enzymatic activity is a key aspect of their activity assessment because it provides insight into the binding affinity of the fluorescent probe to the target protein and its selectivity for cognate proteins.

[0097] The commercial enzyme was pre-incubated with the probe for 30 min, and the phosphatase substrate DiFMUP was added to measure the fluorescence signal of the product. The in vitro inhibition curves of different concentration series of fluorescent probes SHP-PS (1-4) and SHP-099 on SHP2 are shown in Figure 4 .

[0098] Figure 4 The vertical axis is Inhibition-inhibition rate, and the horizontal axis is Concentration-concentration.

[0099] The results showed that each fluorescent probe had strong inhibitory activity against SHP2, but had no inhibitory effect on SHP1 (Table 2), suggesting that it can selectively label SHP2 among homologous proteins.

[0100] Although the inhibitory activity of each fluorescent probe was reduced compared with SHP-099, SHP-PS1 showed the most obvious activity. As the linker lengthened, the activity gradually decreased, reaching approximately 15 times that of SHP-PS4.

[0101] Table 2 IC values of fluorescent probes for inhibition of SHP1 and SHP2 enzymes 50 value

[0102]

[0103]

[0104] In summary, the introduction of fluorophores may inevitably affect the inhibitory activity, but the fluorescent probe provided by the present invention still shows good binding to the target SHP2 and exhibits satisfactory selectivity.

[0105] Example 5 Labeling of SHP2 in living cells

[0106] The present invention conducted cell imaging experiments to determine whether each fluorescent probe can specifically label SHP2 on living cells.

[0107] After the cells were incubated with each fluorescent probe for 20 min, images were taken using an inverted fluorescence microscope without washing.

[0108] Figure 5 Bright-field imaging (L) and fluorescence imaging (R) of different fluorescent probes SHP-PS1 (A), SHP-PS2 (B), SHP-PS3 (C), and SHP-PS4 (D) are shown, where 1 represents the fluorescent probe (2 μmol / L) and 2 represents the fluorescent probe (2 μmol / L) + SHP-099 (20 μmol / L); the scale bar is 50 μm.

[0109] The results showed that all fluorescent probes could label SHP2 in living cells. Under the competition of SHP-099, the decrease in the fluorescence intensity of each fluorescent probe further proved that each fluorescent probe specifically bound to SHP2, rather than turning on fluorescence due to nonspecific binding with other proteins.

[0110] Moreover, under the same conditions, the fluorescence properties of the four probes were different, and SHP-PS2 maintained good SHP2 inhibitory activity (IC 50 =2.88±0.17μmol / L) and environmental sensitivity, and its performance in cell imaging was the most outstanding, showing the prospect of further research.

[0111] In addition, although SHP-PS1 maintained the best SHP2 inhibitory activity (IC 50 =2.10±0.07μmol / L) and is most sensitive to environmental changes, but Figure 5 The results showed that it had the worst imaging effect in living cells compared to the other three fluorescent probes, presumably due to the short linker of SHP-PS1, which prevented the fluorophore from fully entering the hydrophobic environment of the binding site. However, SHP-PS3 and SHP-PS4 also showed satisfactory imaging effects.

[0112] As described above, the various fluorescent probes provided by the present invention can specifically label SHP2, thereby achieving the localization and tracking of SHP2 in biological systems.

[0113] Example 6 Flow cytometry assay to determine the binding of fluorescent probes to SHP2 in living cells

[0114] Flow cytometry experiments were used to study the labeling effect of the probe on SHP2 in living cells.

[0115] like Figure 6 As shown in the figure, red: control, blue: fluorescent probe SHP-PS (1-4) only, yellow: fluorescent probe SHP-PS (1-4) + SHP-099 containing competitive inhibitor (SHP-099).

[0116] Figure 6 As can be seen in the figure, the fluorescence intensity detected increased significantly after the fluorescent probe was incubated with living cells. In addition, under competition with SHP-099 at a concentration of 10 times, the histogram showed a significant left shift, indicating that the cell fluorescence intensity decreased, indicating that SHP-099 inhibited the binding of the fluorescent probe to SHP2.

[0117] Flow cytometry experiments further confirmed that the fluorescent probe prepared by the present invention specifically labels SHP2 in living cells.

[0118] Example 7 Verification of Selective Opening of Probe

[0119] In the present invention, an environment-sensitive switch is designed for the fluorescent probe, which is activated when bound to the target and releases strong fluorescence. The present invention also verifies the switching properties of the fluorescent probe from the perspective of proteins and living cells.

[0120] Figure 7 Figure 2 shows the fluorescence on-characteristics of SHP-PS2 after incubation with different concentrations of SHP2 protein or living cells for 30 min at 37°C. (A) Fluorescence emission spectrum of the fluorescent probe containing SHP2 protein; (B) Fluorescence intensity of the fluorescent probe containing SHP2 protein at the maximum emission wavelength; (C, D) Fluorescence intensity of the fluorescent probe containing living cells at the maximum emission wavelength. Wavelength represents wavelength, Concentration represents concentration, and Cell number represents cell number.

[0121] Figure 7 As shown in the figure, after the fluorescent probe SHP-PS2 was incubated with SHP2 protein, the fluorescence intensity increased significantly. As the protein concentration gradually increased, the fluorescence intensity reached saturation ( Figure 7 Middle A, Figure 7 Middle B). Consistent with previous imaging results, probes SHP-PS2, SHP-PS3, and SHP-PS4 exhibited good switching performance, while SHP-PS1 performed poorly, with weak fluorescence release.

[0122] The present invention verifies the switching characteristics by incubating with living cells ( Figure 7 Middle C, Figure 7 D) and reached a consistent conclusion.

[0123] In general, the environmentally sensitive fluorescent switch designed in the present invention is well implemented, and its performance is greatly affected by the length of the connector.

[0124] Example 8 Selective Determination of Fluorescent Probes for SHP2

[0125] The present invention selects bovine serum albumin (BSA), papain and trypsin as representatives and compares them with SHP2 protein to test the selectivity of the fluorescent probe, thereby verifying whether it may nonspecifically bind to other cellular components, and other proteins are present at twice the concentration of SHP2 protein.

[0126] Figure 8 The graph shows the fluorescence intensity of different proteins and fluorescent probes (1 μmol / L) after incubation for 30 min at 37°C. The blank represents the fluorescent probe solution without protein.

[0127] like Figure 8 As shown, the fluorescence generated by the three fluorescent probes after incubation with bovine serum albumin (BSA), papain, and trypsin was significantly weaker than that generated after incubation with SHP2 (P<0.001). In the presence of SHP-099, competition for binding sites prevented the fluorescent probes from entering the hydrophobic environment to activate the fluorescent switch, resulting in a significant decrease in the fluorescence intensity of the system (P<0.01). Therefore, each fluorescent probe has good selectivity for SHP2, with minimal nonspecific binding.

[0128] Example 9 Fluorescence polarization experiment to determine the affinity of the fluorescent probe to the SHP2 protein

[0129] When a fluorescent probe is in a free state, the molecule rotates or flips freely, resulting in a low fluorescence polarization value. After binding to the target protein, its free movement is restricted, resulting in a slower rotation or flip rate, thus generating strong fluorescence polarization. Fluorescence polarization can not only characterize the affinity of a fluorescent molecule for its target, but can also be further used to establish competitive fluorescence screening methods.

[0130] After incubating SHP2 protein with probes of different concentrations, the fluorescence polarization value increased with the increase of probe concentration and gradually reached a plateau. d The values were calculated using Graphpad Prism 7.00.

[0131] K of SHP-PS1 and SHP-PS2 dThe values were between 1 and 2 μmol / L, and the K d The values were 5.7 μmol / L and 11.2 μmol / L ( Figure 9 ), and their affinity strength was well correlated with the inhibition of SHP2 enzyme activity in vitro. Fluorescence polarization experiments further demonstrated that each fluorescent probe bound well to the SHP2 protein.

[0132] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A fluorescent probe targeting SHP2, characterized in that: The fluorescent probe is SHP-PS2, and the fluorescent probe has the following structure: ； Among them, n=5.

2. Use of the fluorescent probe targeting SHP2 as claimed in claim 1 in the preparation of a SHP2 detection reagent.

Images