A probe molecule for pulling down GSTO1, and its preparation method and application

Abstract

The present invention discloses a probe molecule for GSTO1 pulldown, its preparation method, and application. By utilizing the high nucleophilicity of the cysteine ​​residue in the GSTO1 active center, the probe molecule covalently reacts with the sulfhydryl group to stably pull down the GSTO1 protein. The inhibitor's affinity for GSTO1 in living cells was also tested using a competitive pulldown method. The preparation method comprises the following steps: 2-amino-6-hydroxybenzothiazole (Compound II) is reacted with a haloalkyne to obtain a benzothiazole compound (Compound III); this compound is then reacted with a haloacetyl chloride to obtain a probe molecule (Compound I) capable of pulling down GSTO1. This route is simple to operate, offers a stable process, and significantly reduces the use of active, toxic, and hazardous reagents.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis, and in particular relates to a probe molecule for pulling down GSTO1, a preparation method thereof, and an application thereof. Background Art

[0002] Glutathione S-transferases (GSTs) are a class of proteins widely present in important organelles such as the cytoplasm and mitochondria. In addition to detoxification through glutathione conjugation, GSTs also play a key role in the synthesis and metabolism of endogenous compounds, redox homeostasis, and cell signaling. GSTs can be divided into seven categories. Among them, atypical cytoplasmic glutathione S-transferases, represented by GSTO1, are unique in both structure and function. In recent years, they have been shown to be highly expressed in several cancers and associated with chemotherapy resistance. In addition, GSTO1 is involved in regulating multiple intracellular signaling pathways and is closely related to Alzheimer's disease, Parkinson's disease, chronic obstructive pulmonary disease, and the occurrence of inflammation. Therefore, GSTO1 is an important drug target and biomarker, and research on the function and properties of GSTO1 is crucial for drug development for related indications.

[0003] The catalytically active center of GSTO1 contains a highly nucleophilic cysteine ​​residue, and corresponding probes targeting this active center have been developed for scientific research. Among existing probes, the most commonly used is 5-chloromethylfluorescein diacetate (CMFDA), a cell tracer that has been shown to irreversibly bind to GSTO1. This allows for fluorescent labeling of recombinant GSTO1 proteins and characterization of competitive binding of inhibitors at the gel level [Nat Commun. 2016, 7, 1308]. CMFDA can also label endogenous GSTO1. However, due to the diversity and complexity of endogenous proteins, CMFDA also labels other cytoplasmic proteins, making it difficult to determine the identity of the fluorescent signal, potentially leading to false positive binding signals. Furthermore, this probe is limited to fluorescence imaging and cannot be used to isolate and identify the target protein through pull-down, which limits accurate identification and quantification of the target protein. Pull-down experiments are of great significance in biological research. By pulling down the target protein, we can further characterize the abundance of a certain protein in the indication, identify the interaction network of the target protein, infer its function and regulatory pathway, and study the relationship between the drug and the target, etc., providing an important biological basis for subsequent drug development and clinical disease treatment. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a probe molecule for pulling down GSTO1 and a preparation method and application thereof.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] A probe molecule for pulling down GSTO1, wherein the probe molecule has a structure shown in Formula I:

[0007]

[0008] The probe molecule for pulling down GSTO1 comprises the following steps:

[0009]

[0010] In the step of preparing compound III from compound II, the halogenated alkyne is 6-iodohexyne, 6-chlorohexyne or 6-bromohexyne, and the base is potassium carbonate or sodium carbonate.

[0011] In the preparation of compound III from compound II, the molar ratio of compound II:halogenated alkyne:base is in the range of 1:1-1.05:3-5.

[0012] In the preparation of compound III from compound II, the reaction solvent is acetone or tetrahydrofuran, the reaction is heated under reflux, and the reaction time is 18 to 24 hours.

[0013] In the preparation of compound I from compound III, the haloacetyl chloride is chloroacetyl chloride, the acid is trifluoroacetic acid or acetic acid, preferably trifluoroacetic acid, and the molar ratio of compound III: haloacetyl chloride: acid is in the range of 1:1-1.1:1-1.1.

[0014] In the preparation of compound I from compound III, the reaction solvent is acetone or tetrahydrofuran, the reaction temperature is 15° C. to 25° C., and the reaction time is 0.5 to 3 h.

[0015] An application of a probe molecule for pulling down GSTO1, wherein the probe molecule can be used to pull down GSTO1 protein from cells and tissues.

[0016] The invention discloses an application of a probe molecule for pulling down GSTO1, wherein the probe molecule can be used to detect and quantify the abundance of GSTO1 protein in cells and tissues.

[0017] The invention discloses an application of a probe molecule for pulling down GSTO1. The probe molecule can be used to screen GSTO1 inhibitors, verify the inhibitory activity of small molecule compounds on GSTO1, test the affinity of small molecule compounds with GSTO1, or detect GSTO1 protein activity.

[0018] Beneficial effects: The present invention discloses a probe molecule for pulling down GSTO1, and a preparation method and application thereof. The high nucleophilic ability of the cysteine ​​residue in the active center of GSTO1 is utilized, and the covalent reactivity of the probe molecule to the sulfhydryl group is used to stably pull down the GSTO1 protein. The affinity of the inhibitor molecule for GSTO1 in living cells is also tested by a competitive pull-down method. The inhibition of the protein band pulled down by the probe molecule by the inhibitor is detected by protein blotting, indicating that the probe has good application prospects as a tool molecule. In the future, it can be used to screen GSTO1 regulator molecules, detect the abundance of GSTO1 protein in quantitative cells and tissues, clarify the pharmacodynamic mechanism of GSTO1 regulators (upregulating or downregulating GSTO1) and the relationship between efficacy and target protein abundance, and guide patient selection in clinical studies and clinical data group analysis. The preparation method includes the following steps: using 2-amino-6-hydroxybenzothiazole (compound II) as a raw material, reacting it with a halogenated alkyne to obtain a benzothiazole compound (compound III); then reacting it with a halogenated acetyl chloride to obtain a probe molecule (compound I) that can pull down GSTO1. This route is simple to operate, has a stable process, greatly reduces the use of active and toxic and hazardous reagents, and is suitable for large-scale industrial production.

[0019] The abbreviations of the reaction reagents involved in the instructions are as follows:

[0020] K2CO3: potassium carbonate;

[0021] H2O: water. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the H NMR spectrum of compound I;

[0023] Figure 2 The results of different concentrations of probes in Experimental Example 1 pulling down GSTO1 in Hela cells;

[0024] Figure 3 The results of Experimental Example 1 are as follows: different concentrations of probes were used to pull down GSTO1 in HCT116 cells;

[0025] Figure 4 This is the result of Experimental Example 2 where the GSTO1 inhibitor C1-27 inhibited the probe from pulling down GSTO1 in Hela cells. DETAILED DESCRIPTION

[0026] The present invention will be further illustrated below with reference to specific examples. These examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0027] Example 1

[0028] Synthesis of Compound I:

[0029] Compound II (2-amino-6-hydroxybenzothiazole) (2.00 g, 12.03 mmol, 1.00 eq), iodohexyne (2.63 g, 12.64 mmol, 1.05 eq), KCO (7.82 g, 56.56 mmol, 4.70 eq), and acetone (30 mL) were added to a 100 mL single-necked flask and stirred. The mixture was heated to 65°C and refluxed for 18 hours, resulting in the reaction. The reaction mixture was cooled to room temperature and concentrated to dryness. Purification by column chromatography (PE:EA = 2:1) afforded intermediate compound III (860 mg of an off-white solid, yield: 29.05%).

[0030] The intermediate compound III (425 mg, 1.73 mmol, 1.00 eq), trifluoroacetic acid (192 mg, 1.90 mmol, 1.10 eq) and tetrahydrofuran (5 mL) were added to a 500 mL single-necked bottle and stirred. Chloroacetyl chloride (215 mg, 1.90 mmol, 1.10 eq) was added dropwise at 0°C. Stirring at room temperature for 1 hour, the reaction was completed. The reaction solution was concentrated to dryness, H2O (20 mL) was added and stirred. Filtered, the solid was collected, and refluxed with ethanol (8 mL) for 15 minutes. Filtered, the solid was collected, and refluxed with methanol (8 mL × 2) for 15 minutes. Filtered, the solid was collected, and dried under reduced pressure. The probe molecule compound I (305 mg of white solid, yield: 71.94%) was obtained.

[0031] Probe molecule compound I 1 H NMR spectrum Figure 1 . 1 H NMR (400MHz, DMSO-d6) δ12.58 (s, 1H), 7.66 (d, J = 8.8Hz, 1H), 7.59 (d, J = 2.5Hz, 1H), 7.05 (dd, J = 8.8, 2.6Hz, 1H), 4.44 ( s, 2H), 4.04 (t, J=6.4Hz, 2H), 2.79 (t, J=2.7Hz, 1H), 2.25 (td, J=7.1, 2.7Hz, 2H), 1.89-1.77 (m, 2H), 1.68-1.57 (m, 2H).

[0032] LCMS calculation for C 14 H 25 N5O([M+H] + ):323.0, found:323.1.

[0033] Example 2

[0034] Synthesis of Compound I:

[0035] Compound II (2-amino-6-hydroxybenzothiazole) (2.00 g, 12.03 mmol, 1.00 eq), iodohexyne (2.50 g, 12.03 mmol, 1.0 eq), KCO (4.98 g, 36.09 mmol, 3.0 eq), and acetone (30 mL) were added to a 100 mL single-necked flask and stirred. The mixture was heated to 65°C and refluxed for 18 hours, resulting in completion of the reaction. The reaction mixture was cooled to room temperature and concentrated to dryness. Purification by column chromatography (PE:EA = 2:1) afforded intermediate compound III (838 mg off-white solid, yield: 28.31%).

[0036] The intermediate compound III (425 mg, 1.73 mmol, 1.00 eq), trifluoroacetic acid (197 mg, 1.73 mmol, 1.0 eq) and tetrahydrofuran (5 mL) were added to a 500 mL single-necked bottle and stirred. Chloroacetyl chloride (195 mg, 1.73 mmol, 1.0 eq) was added dropwise at 0°C. Stirring at room temperature for 1 hour, the reaction was completed. The reaction solution was concentrated to dryness, H2O (20 mL) was added and stirred. Filtered, the solid was collected, and refluxed with ethanol (8 mL) for 15 minutes. Filtered, the solid was collected, and refluxed with methanol (8 mL × 2) for 15 minutes. Filtered, the solid was collected, and dried under reduced pressure. The probe molecule compound I (303 mg of white solid, yield: 71.47%) was obtained.

[0037] Example 3

[0038] Synthesis of Compound I:

[0039] Compound II (2-amino-6-hydroxybenzothiazole) (2.00 g, 12.03 mmol, 1.00 eq), iodohexyne (2.50 g, 12.03 mmol, 1.0 eq), KCO (8.30 g, 60.15 mmol, 5.0 eq), and tetrahydrofuran (30 mL) were added to a 100 mL single-necked flask and stirred. The mixture was heated to 70°C and refluxed for 18 hours, resulting in completion of the reaction. The reaction mixture was cooled to room temperature and concentrated to dryness. Purification by column chromatography (PE:EA = 2:1) afforded intermediate compound III (874 mg off-white solid, yield: 29.52%).

[0040] The intermediate compound III (425 mg, 1.73 mmol, 1.00 eq), trifluoroacetic acid (197 mg, 1.73 mmol, 1.0 eq) and tetrahydrofuran (5 mL) were added to a 500 mL single-necked bottle and stirred. Chloroacetyl chloride (195 mg, 1.73 mmol, 1.0 eq) was added dropwise at 0°C. Stirring was carried out at room temperature for 1 hour and the reaction was completed. The reaction solution was concentrated to dryness, H2O (20 mL) was added and stirred. Filtered, the solid was collected, and refluxed with ethanol (8 mL) for 15 minutes. Filtered, the solid was collected, and refluxed with methanol (8 mL × 2) for 15 minutes. Filtered, the solid was collected, and dried under reduced pressure. The probe molecule compound I (305 mg of white solid, yield: 71.94%) was obtained.

[0041] Test Example 1

[0042] Evaluation of the pull-down effect of probe molecules on target protein GSTO1 in living cells:

[0043] Table 1 Preparation of cell lines, culture media and test compounds

[0044] cell lines culture medium HCT116 McCoy's 5a + 10% FBS Hela DMEM + 10% FBS

[0045] As shown in Table 1, the cell lines to be tested were cultured in a medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin (see table for ingredients) at 37°C in a cell culture incubator with 5% CO2. The medium was changed every two days. After cells reached 80% confluency, they were trypsinized and passaged to maintain cells in a vigorous logarithmic growth phase.

[0046] Detection method:

[0047] HeLa cells in the logarithmic growth phase were 5*10 6 Seed cells per dish in a 10 cm cell culture dish and culture for 24 hours. Wash the cells with PBS and serum-free medium. Prepare serum-free medium containing different concentrations of probe and add it to the culture dish. Incubate the cells in an incubator for another hour. Then, place the cell culture dish on ice and wash the cells twice with cold PBS. Scrape the cells with PBS and collect them.

[0048] The cells were added to a cell lysis buffer [PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), 0.1% Benzonase (Beyotime)] and sonicated before high-speed centrifugation (20,000 g, 30 minutes) at 4°C. The supernatant was collected and the concentration was adjusted to 2 mg / mL using the cell lysis buffer. To 500 μL of the protein solution, a final concentration of 200 μM biotin azide, 2.5 mM sodium ascorbate (Sigma-Aldrich), 25 mM BTTAA (CONFLUORE), and 12.5 mM CuSO₄ (Innochem) were added at room temperature and allowed to react for 1 hour. After completion of the reaction, the protein was recovered by methanol-chloroform precipitation. After resuspension, 300 μL of streptavidin-coated magnetic beads were added and incubated at room temperature for 3 hours to enrich the probe-labeled proteins. After enrichment, 1X loading buffer was added and incubated at 95°C for 20 minutes. The protein sample was magnetically separated and separated by SDS-PAGE. The protein signal was then transferred to a NC membrane by western blotting. After transfer, the NC membrane was soaked in Ponceau red staining solution and the Ponceau red signal was read. The NC membrane was placed in 10 mL of 5% BSA / TBST and incubated at room temperature for 1 hour. The target protein antibody GSTO1 Polyclonal Antibody (Proteintech) was incubated overnight at 4°C. Finally, a secondary antibody (Alexa Fluor 488-labeled Goat Anti-Rabbit IgG, Beyotime) was incubated for 1 hour. The secondary antibody fluorescence signal was scanned using ChemiDoc imaging.

[0049] The results of the experiment in which the probe pulled down GSTO1 protein in Hela and HCT116 cells are as follows: Figure 2 、 3 As shown, the GSTO1 protein was stably pulled down by covalent reaction of the probe molecule with the cysteine ​​residue in the active center of the GSTO1 protein; the protein band intensity before and after enrichment was compared by protein blotting, indicating that the probe can efficiently pull down the GSTO1 protein.

[0050] Test Example 2

[0051] Assessment of target protein GSTO1 occupancy activity in living cells:

[0052] Table 2 Preparation of cell lines, culture media and test compounds

[0053] cell lines culture medium Hela DMEM + 10% FBS

[0054] As shown in Table 2, HeLa cells were cultured in a medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin (see table for ingredients) at 37°C in a cell culture incubator with 5% CO2. The medium was changed every two days. After cells reached 80% confluency, they were trypsinized and passaged to maintain the cells in a favorable logarithmic growth phase.

[0055] Detection method:

[0056] HCT116 cells in the logarithmic growth phase were expressed as 1*10 7 Cells were seeded into 10 cm cell culture dishes and cultured for 24 hours. The cells were washed with PBS and serum-free medium. Serum-free medium containing the GSTO1 inhibitor C1-27 was prepared and added to the culture dishes. The cells were cultured in an incubator for another 2 hours. The probe was then added to a final concentration of 100 μM and cultured for another 1 hour. The cell culture dishes were then placed on ice and the cells were washed twice with cold PBS. PBS was then added to scrape the cells and collect them.

[0057] The cells were added to a cell lysis buffer [PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), 0.1% Benzonase (Beyotime)] and sonicated before high-speed centrifugation (20,000 g, 30 minutes) at 4°C. The supernatant was collected and the concentration was adjusted to 2 mg / mL using the cell lysis buffer. To 500 μL of the protein solution, a final concentration of 200 μM biotin azide, 2.5 mM sodium ascorbate (Sigma-Aldrich), 25 mM BTTAA (CONFLUORE), and 12.5 mM CuSO₄ (Innochem) were added at room temperature and allowed to react for 1 hour. After completion of the reaction, the protein was recovered by methanol-chloroform precipitation. After resuspension, 300 μL of streptavidin-coated magnetic beads were added and incubated at room temperature for 3 hours to enrich the probe-labeled proteins. After enrichment, 1X loading buffer was added and incubated at 95°C for 20 minutes. The protein sample was magnetically separated and separated by SDS-PAGE. The protein signal was then transferred to a NC membrane by western blotting. After transfer, the NC membrane was soaked in Ponceau red staining solution and the Ponceau red signal was read. The NC membrane was placed in 10 mL of 5% BSA / TBST and incubated at room temperature for 1 hour. The target protein antibody GSTO1 Polyclonal Antibody (Proteintech) was incubated overnight at 4°C. Finally, a secondary antibody (Alexa Fluor 488-labeled Goat Anti-Rabbit IgG, Beyotime) was incubated for 1 hour. The secondary antibody fluorescence signal was scanned using ChemiDoc imaging.

[0058] The inhibitor molecule C1-27 was tested by compound competition probe to pull down GTSO1 protein: (CAS: 568544-03-6) affinity for GSTO1 in living cells, see Figure 4 .

[0059] Inhibition rate (%) = 100-(target band signal of sample group / target band signal of blank group)*100

[0060] Table 3 Inhibition rate of compounds on GSTO1 pull-down

[0061]

[0062] Protein blotting was used to detect the compounds that inhibited the probe molecule from pulling down the GSTO1 protein. The higher the inhibition rate, the weaker the protein band intensity (see Table 3 for inhibition rate). The results showed that the probe can be used as a tool molecule for GSTO1 regulator compound screening, activity verification, affinity testing, or testing GSTO1 protein activity.

[0063] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of this application in detail. It should be understood that the above are only specific implementation methods of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of this application should be included in the scope of protection of this application.

Claims

1. A probe molecule for pulling down GSTO1, wherein: It has the structure shown in formula I:

2. A method for preparing a probe molecule for pulling down GSTO1, for preparing the probe molecule for pulling down GSTO1 according to claim 1, wherein: The steps include:

3. The preparation method according to claim 2, characterized in that In the step of preparing compound III from compound II, the halogenated alkyne is 6-iodohexyne, 6-chlorohexyne or 6-bromohexyne, and the base is potassium carbonate or sodium carbonate.

4. The preparation method according to claim 2 or 3, characterized in that In the preparation of compound III from compound II, the molar ratio of compound II:halogenated alkyne:base is in the range of 1:1-1.05:3-5.

5. The preparation method according to claim 2 or 3, characterized in that In the preparation of compound III from compound II, the reaction solvent is acetone or tetrahydrofuran, the reaction is heated under reflux, and the reaction time is 18 to 24 hours.

6. The preparation method according to claim 2, characterized in that In the preparation of compound I from compound III, the haloacetyl chloride is chloroacetyl chloride, the acid is trifluoroacetic acid or acetic acid, and the molar ratio of compound III: haloacetyl chloride: acid is in the range of 1:1 to 1.1:1 to 1.

1.

7. The preparation method according to claim 2 or 6, characterized in that: In the preparation of compound I from compound III, the reaction solvent is acetone or tetrahydrofuran, the reaction temperature is 15°C to 25°C, and the reaction time is 0.5 to 3 hours.

8. A use of a probe molecule for pulling down GSTO1 according to any one of claims 1 to 7, characterized in that: The probe molecule can be used to pull down GSTO1 protein from cells and tissues.

9. A use of a probe molecule for pulling down GSTO1 according to any one of claims 1 to 7, characterized in that: The probe molecule can be used to detect and quantify the abundance of GSTO1 protein in cells and tissues.

10. A use of a probe molecule for pulling down GSTO1 according to any one of claims 1 to 7, characterized in that: The probe molecule can be used to screen GSTO1 inhibitors, verify the inhibitory activity of small molecule compounds on GSTO1, test the affinity of small molecule compounds to GSTO1, or detect GSTO1 protein activity.

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