ABPP-based anti-inflammatory drug 18 beta-glycyrrhetinic acid active molecular probe and synthesis and application thereof

Abstract

The invention discloses an active molecular probe synthesized by taking 18 beta-glycyrrhetinic acid as a parent compound as well as a preparation method and application of the active molecular probe. The compound has the same anti-inflammatory activity as 18 beta-glycyrrhetinic acid, and can significantly reduce the expression of vascular endothelial cells (PIEC) COX-2, IL-6 and TNF-alpha induced by pasteurella multocida HB03. Besides, the active molecular probe has an alkynyl group, and can be connected with a reporter group through a click chemical reaction, so that localization of the 18beta-glycyrrhetinic acid medicine in cells and analysis and identification of acting target protein are realized.

Description

Technical Field

[0001] The present invention belongs to the field of chemical biology, and in particular, relates to an ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe and its synthesis and application. Background Art

[0002] 18β-glycyrrhetinic acid is a monomeric small molecule drug extracted from licorice. Its chemical structure is similar to that of steroid hormones. It has anti-inflammatory, anti-ulcer, antibacterial, antioxidant, immunomodulatory, cardiovascular and neuroprotective activities. In terms of anti-inflammatory, 18β-glycyrrhetinic acid protects small intestinal epithelial cells from LPS-induced cellular inflammatory damage by regulating COX-2 expression; inhibits vascular endothelial inflammatory damage induced by porcine Pasteurella multocida through PARP-1 / NLRP3 and NF-κB signaling pathways; inhibits Staphylococcus aureus-induced RAW264.7 cell inflammation by inhibiting the activation of HMGB1 / NF-κB; and has a protective effect on gastric mucosal inflammation in Mongolian gerbils infected with Helicobacter pylori by reducing the expression of inflammatory-related cytokines (IL-1β, TNF-α, COX-2 and i-NOS).

[0003] Activity-based protein profiling (ABPP) is an emerging proteomics technology that integrates chemistry and biology. It uses probe modification of small molecules to fish for targets and then uses proteomics detection to accurately explore the protein targets of small molecule compounds in living cells. It has important research and application value in disease and drug research.

[0004] Classic ABPs consist of two parts: a reactive group and a reporter group. The reactive group is usually the parent structure of a small drug molecule, which can covalently bind to the catalytic center of the target protein, thereby achieving the purpose of activity-based labeling. The reporter group is used for tracing to facilitate detection, such as fluorescent labeling, isotope labeling, and biotin labeling. In recent years, with the development of bioorthogonal reactions, the reporter group in active probe molecules has gradually been replaced by inert bioorthogonal reactive groups such as alkynyl and azide. After the probe is labeled, a click chemistry reaction is used to connect a reporter group such as fluorescent or biotin to the probe. Such a design can greatly reduce the overall steric hindrance of the active molecular probe, which is beneficial for the live labeling of the probe in cells and tissues, and also simplifies the steps of probe synthesis. Summary of the invention

[0005] The purpose of the present invention is to design and synthesize an ABPP-based active molecular probe for the anti-inflammatory drug 18β-glycyrrhetinic acid in order to solve the above problems. The probe not only retains the anti-inflammatory activity of the parent compound 18β-glycyrrhetinic acid, but also has an acetylene group that 18β-glycyrrhetinic acid does not have. It can be connected to a reporter group through a click chemistry reaction to achieve the localization of the 18β-glycyrrhetinic acid drug in the cell and the confirmation of the target protein, providing a powerful tool for the study of the anti-inflammatory targets and molecular mechanisms of the 18β-glycyrrhetinic acid drug.

[0006] The technical solution adopted by the present invention is:

[0007] On the one hand, an ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid activity molecular probe has the structure shown below:

[0008]

[0009] In the above structure, the alkynyl group connected to the rightmost end serves as a reporter group in the active molecular probe.

[0010] On the other hand, the present invention also provides a method for synthesizing an active molecular probe of an anti-inflammatory drug 18β-glycyrrhetinic acid based on ABPP, the steps of which are as follows:

[0011] Dissolve 18β-glycyrrhetinic acid in an organic solvent and stir until it is clear; then add 3-(3-alkyne-1-butyl)-3-(2-iodoethyl)-3H-diaziridine, add a base after a period of reaction, and after the reaction is complete, perform post-treatment to obtain the target compound;

[0012]

[0013] Furthermore, the organic solvent is selected from one or more of methanol, ethanol, dichloromethane, chloroform, acetone and DMF.

[0014] Furthermore, the base is selected from: one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

[0015] Furthermore, the reaction was carried out at room temperature in the dark overnight, and the reaction progress was monitored by TLC.

[0016] Furthermore, the post-treatment step includes: after the reaction is complete, adding saturated brine under ice bath to terminate the reaction; transferring to a separatory funnel, washing the round-bottom flask with a small amount of ethyl acetate, adding ethyl acetate to extract 3 times, and combining the organic phases; adding saturated brine to wash the organic layer, repeating 3 times, combining the organic phases, adding anhydrous sodium sulfate to dry and remove water, and removing the organic phase by reduced pressure distillation to obtain the target compound.

[0017] Furthermore, the synthesis method of the active molecular probe of the anti-inflammatory drug 18β-glycyrrhetinic acid based on ABPP comprises the following steps:

[0018] Accurately weigh 47.1 mg of 18β-glycyrrhetinic acid and dissolve it in 5 mL of N-N-dimethylformamide (DMF), and stir until clear. Then add 32.2 mg of 3-(3-alkyne-1-butyl)-3-(2-iodoethyl)-3H-diaziridine, react for 10 minutes, add 27.6 mg of potassium carbonate, react overnight at room temperature in the dark, monitor the reaction progress by TLC, and after the reaction is complete, add 5 mL of saturated brine under ice bath to terminate the reaction. Transfer to a separatory funnel, wash the round-bottom flask with a small amount of ethyl acetate, add 5 mL of ethyl acetate to extract 3 times, and combine the organic phases. Add 10 mL of saturated brine to wash the organic layer, repeat 3 times, combine the organic phases, add anhydrous sodium sulfate to dry and remove water, and remove the organic phase by vacuum distillation to obtain the target compound (GAL3).

[0019] In another aspect, the present invention also provides an application of an ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe in the preparation of anti-inflammatory drugs.

[0020] Furthermore: after the needle is labeled in living cells, the part with the same structure as the parent compound 18β-glycyrrhetinic acid can be used as a reactive group to selectively bind to the active center of a certain type of enzyme in the intracellular proteome and react with the affinity amino acids that perform important catalytic functions, thereby covalently labeling the probe molecule on the target protein; then, a fluorescent group or a biotin group can be connected to the probe through a click chemistry bioorthogonal reaction;

[0021] When attached with a fluorescent group, the proteome labeled by the probe molecule can be separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and show a specific fluorescent signal;

[0022] When the biotin group is attached, the labeled protein can be specifically enriched by avidin and then analyzed by biomacromolecule mass spectrometry to determine the labeled protein components.

[0023] Furthermore, the click chemistry bioorthogonal reaction is a copper-catalyzed azide-alkynyl cycloaddition reaction.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The present invention synthesizes an ABPP-based active molecular probe of the anti-inflammatory drug 18β-glycyrrhetinic acid, which introduces a bioorthogonal reaction group alkynyl into the structure of the anti-inflammatory drug 18β-glycyrrhetinic acid. It not only retains the anti-inflammatory activity of 18β-glycyrrhetinic acid, but also can be connected to a reporter group such as fluorescein or biotin through a click chemistry reaction after interacting with living cells, thereby achieving the localization of the drug in the cell and the labeling and identification of the target protein, providing a powerful tool for the study of the anti-inflammatory target and mechanism of action of 18β-glycyrrhetinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 , Mass spectrum of GAL3, an active molecular probe for the anti-inflammatory drug 18β-glycyrrhetinic acid based on ABPP.

[0027] Figure 2 , hydrogen spectrum of GAL3, an active molecular probe for the anti-inflammatory drug 18β-glycyrrhetinic acid based on ABPP.

[0028] Figure 3 , carbon spectrum of the ABPP-based active molecular probe GAL3 for the anti-inflammatory drug 18β-glycyrrhetinic acid.

[0029] Figure 4 , the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe and the effects of the parent compound 18β-glycyrrhetinic acid on the expression of COX-2, IL-6 and TNF-α in PIEC cells induced by Pasteurella multocida HB03, where GA is 18β-glycyrrhetinic acid and GAL3 is the active molecular probe of 18β-glycyrrhetinic acid. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0031] The technical solution of the present invention is further explained below in conjunction with implementation cases.

[0032] A method for synthesizing an active molecular probe of an anti-inflammatory drug 18β-glycyrrhetinic acid based on ABPP, wherein the molecular probe has the following structure:

[0033]

[0034] The specific steps are as follows:

[0035] Accurately weigh 47.1 mg of 18β-glycyrrhetinic acid and dissolve it in 5 mL of N-N-dimethylformamide (DMF), and stir until clear. Then add 32.2 mg of 3-(3-alkyne-1-butyl)-3-(2-iodoethyl)-3H-diaziridine, react for 10 minutes, add 27.6 mg of potassium carbonate, react overnight at room temperature in the dark, monitor the reaction progress by TLC, and after the reaction is complete, add 5 mL of saturated brine under ice bath to terminate the reaction. Transfer to a separatory funnel, wash the round-bottom flask with a small amount of ethyl acetate, add 5 mL of ethyl acetate to extract 3 times, and combine the organic phases. Add 10 mL of saturated brine to wash the organic layer, repeat 3 times, combine the organic phases, add anhydrous sodium sulfate to dry and remove water, and remove the organic phase by vacuum distillation to obtain the target compound (GAL3).

[0036] The specific reaction route is as follows:

[0037]

[0038] In vitro anti-inflammatory activity assay

[0039] PIEC cells were cultured in RPMI complete medium and used for experiments when the cells entered the logarithmic growth phase. The cell density was adjusted to 2×10 6 / mL, inoculated in 6-well plates, and incubated at 37°C, 5% CO 2 The culture was continued in the incubator for 12 h. Based on the results of the Pm growth curve, a 12-h Pm bacterial solution (1×10 10 CFU / mL), and calculate the volume of bacterial solution when the multiplicity of infection MOI = 100.

[0040] Place the bacterial solution in a 1.5 mL centrifuge tube and centrifuge at 8000 g for 5 min to set aside the precipitate; wash three times with 1 mL of PBS to rinse the remaining bacterial culture medium, and resuspend the bacteria in an appropriate 5% RPMI 1640 culture medium to obtain a suitable number of infected cells; add the prepared bacterial infection solution to a 6-well plate and shake it left and right to mix, 5% CO 2 , cultured at 37°C for 1 h, then discarded the bacterial infection solution, rinsed the cells twice with preheated PBS, added 18β-glycyrrhetinic acid and GAL3 at final concentrations of 10, 20, and 40 μg / mL, and 5% CO 2, cultured at 37℃ for 12h; washed the cells in the six-well plate twice with pre-cooled PBS, added 200μL cell lysis solution to each well, placed on ice for 20min, scraped the lysed cells with a cell scraper, and placed in a 1.5mL centrifuge tube. Ultrasonic cell disruptor was used to ultrasonically disrupt the cells (40% power, ultrasonic 30s, ultrasonic 3 times, 10s each time, 15s interval in between). BCA was used to determine the protein concentration, 45μg cell protein in each group was denatured, separated on 12.5% ​​SDS-PAGE gel, and transferred to PVDF membrane. The membrane was then blocked with 5% BSA blocking buffer for 2h, washed, and then incubated with the primary antibody at 4℃ overnight. The membrane was washed 3 times with TBST and then incubated with the secondary anti-IgG antibody coupled to horseradish peroxidase at room temperature for 1h. Exposure and development were performed using a color development kit.

[0041] The results of in vitro anti-inflammatory activity assay showed that the newly synthesized probe retained the anti-inflammatory activity of the parent compound 18β-glycyrrhizic acid and could significantly reduce the expression of COX-2, IL-6 and TNF-α in PIEC cells induced by Pasteurella multocida HB03.

[0042] In addition, the active molecular probe has an alkynyl group that the parent compound 18β-glycyrrhetinic acid does not have, and can be connected to a reporter group through a click chemistry reaction, thereby achieving the localization of 18β-glycyrrhetinic acid drugs in cells and the analysis and identification of target proteins. Therefore, the probe can be used as an active molecular probe tool to explore the anti-inflammatory targets and mechanisms of action of 18β-glycyrrhetinic acid drugs.

[0043] Target identification: In a cell or tissue environment, since the probe retains the active structural part of 18β-glycyrrhetinic acid, it can specifically bind to its potential protein target, and covalently bind to the active site of the target protein through the reactive group on the probe. Then, using the detection label on the probe (such as fluorescent label, biotin label, etc.), the specific protein target that interacts with 18β-glycyrrhetinic acid can be identified through corresponding detection techniques (fluorescence microscopy observation, avidin-biotin system enrichment combined with mass spectrometry analysis, etc.), thereby further clarifying the molecular mechanism of 18β-glycyrrhetinic acid in exerting its pharmacological effects.

[0044] Drug screening and optimization: In the process of drug development, the ABPP-based 18β-glycyrrhetinic acid active molecular probe can be used to screen compounds with similar targets or mechanisms of action as 18β-glycyrrhetinic acid. The probe is mixed with a series of compounds to be screened and then incubated with cells or protein samples. If a compound can competitively inhibit the binding of the probe to the target protein, it means that the compound may have similar activity and mechanism of action as 18β-glycyrrhetinic acid, so that the compound can be further studied and optimized, and it is possible to develop more effective drugs or drug combinations.

[0045] Study on drug action mechanism: By observing the changes in the proteins labeled by the probe (such as changes in expression level, changes in activity, etc.), the molecular mechanism by which 18β-glycyrrhetinic acid exerts its pharmacological effects can be inferred.

[0046] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An ABPP-based active molecular probe for anti-inflammatory drug 18β-glycyrrhetinic acid, characterized in that: The molecular probe has the following structure: In the above structure, the alkynyl group connected to the rightmost end serves as a reporter group in the active molecular probe.

2. A method for synthesizing an ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe as claimed in claim 1, characterized in that: Here are the steps: Dissolve 18β-glycyrrhetinic acid in an organic solvent and stir until it is clear; then add 3-(3-alkyne-1-butyl)-3-(2-iodoethyl)-3H-diaziridine, add a base after a period of reaction, and after the reaction is complete, perform post-treatment to obtain the target compound; 3. The method for synthesizing the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe according to claim 2, characterized in that: The organic solvent is selected from one or more of methanol, ethanol, dichloromethane, chloroform, acetone and DMF.

4. The method for synthesizing the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe according to claim 2, characterized in that: The base is selected from: one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

5. The method for synthesizing the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe according to claim 2, characterized in that: The reaction was carried out at room temperature in the dark overnight and the progress of the reaction was monitored by TLC.

6. The method for synthesizing the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe according to claim 2, characterized in that: The post-treatment steps include: after the reaction is complete, adding saturated brine in an ice bath to terminate the reaction; transferring to a separatory funnel, washing the round-bottom flask with a small amount of ethyl acetate, adding ethyl acetate to extract 3 times, and combining the organic phases; adding saturated brine to wash the organic layer, repeating 3 times, combining the organic phases, adding anhydrous sodium sulfate to dry and remove water, and removing the organic phase by reduced pressure distillation to obtain the target compound.

7. Use of the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe as claimed in claim 1 in the preparation of anti-inflammatory drugs.

8. The use of the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe in the preparation of anti-inflammatory drugs according to claim 7, characterized in that: After the needle is labeled in living cells, the part with the same structure as the parent compound 18β-glycyrrhetinic acid can be used as a reactive group to selectively bind to the active center of a certain type of enzyme in the intracellular proteome and react with the affinity amino acids that perform important catalytic functions, thereby covalently labeling the probe molecule on the target protein; then, a fluorescent group or a biotin group can be connected to the probe through a click chemistry bioorthogonal reaction; When attached with a fluorescent group, the proteome labeled by the probe molecule can be separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and show a specific fluorescent signal; When the biotin group is attached, the labeled protein can be specifically enriched by avidin and then analyzed by biomacromolecule mass spectrometry to determine the labeled protein components.

9. The use of the ABPP-based anti-inflammatory drug 18β-glycyrrhetinic acid active molecular probe in the preparation of anti-inflammatory drugs according to claim 8, characterized in that: The click chemistry bioorthogonal reaction is a copper-catalyzed azide-alkynyl cycloaddition reaction.

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