Application of galangin in preparation of ANXA2 targeting drugs

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of galangin in preparation of a targeted ANXA2 medicine. It is verified that galangin can be selectively combined with ANXA2, target ANXA2 in macrophages, promote degradation of ANXA2 in macrophages, reduce release of M1 type macrophage related molecules and increase release of M2 type macrophage related molecules, and has a treatment effect on inflammatory skin diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of galangin in the preparation of drugs targeting ANXA2. Background Art

[0002] Galangin, a natural flavonol compound extracted from Alpinia officinarum, also known as galangin, 3,5,7-trihydroxyflavone, with a CAS number of 548-83-4. A large number of studies have shown that galangin has a variety of biological activities, including anti-mutation, antioxidant, anti-inflammatory, vasodilation, and anti-tumor effects. A large number of studies have shown that galangin has a variety of biological activities and has been found to have a variety of anti-tumor mechanisms, including anti-proliferation, pro-apoptosis, pro-autophagy, and inhibition of tumor cell metastasis. Galangin has a variety of physiological activities such as delaying aging, enhancing the body's defense, and reducing vitiligo symptoms. Currently, there is no report on the use of galangin in the preparation of drugs targeting ANXA2, degrading ANXA2 drugs, and treating psoriasis drugs.

[0003] Annexin A2 (ANXA2) is a membrane-associated protein present in the intracellular and extracellular environments, which plays physiological functions in many tissues and is involved in a variety of pathological processes. In the field of skin diseases, ANXA2 plays a role in the formation of psoriasis, antiphospholipid syndrome, keloid, and scar cancer. ANXA2 is abnormally expressed in a variety of skin diseases and is a potential psoriasis severity grading index, and its role in the pathogenesis of a variety of skin diseases is unclear. Among them, whether ANXA2 affects the skin microenvironment through the interaction with macrophages through pathways such as NF-KB and TLR4, and whether the ANXA2 gene is a susceptible gene for certain autoimmune diseases remain to be solved. In the field of tumor diseases, ANXA2 has been found to exist in tumor cell exosomes and is often highly expressed in tumor cells, thereby affecting the physiological activities of tumor cell adhesion, proliferation, invasion, and migration. Currently, ANXA2 has become one of the important abnormally expressed molecules involved in tumorigenesis in the eyes of researchers, and it has been identified as an important biomarker and a potential target for the treatment of malignant tumors. Summary of the Invention

[0004] The purpose of the present invention is to solve the technical problem that ANXA2, as a key protein in a variety of diseases, lacks relevant targeting means. In this regard, the present invention meets this need in the art by providing the application of galangin in the preparation of drugs targeting ANXA2.

[0005] On the one hand, the present invention relates to the application of galangin in the preparation of drugs targeting ANXA2, and galangin selectively binds to ANXA2.

[0006] Furthermore, in the application provided by the present invention, galangin targets ANXA2 in macrophages.

[0007] On the other hand, the present invention relates to the application of galangin in the preparation of drugs for degrading ANXA2, and galangin promotes the degradation of ANXA2.

[0008] Furthermore, in the application provided by the present invention, galangin targets ANXA2 in macrophages, selectively binds to ANXA2, and promotes the degradation of ANXA2 in macrophages.

[0009] On the other hand, the present invention relates to the application of galangin in the preparation of drugs for improving inflammatory skin diseases.

[0010] Furthermore, in the application provided by the present invention, the inflammatory skin disease is psoriasis.

[0011] Furthermore, in the application provided by the present invention, galangin does not affect the apoptosis of macrophages, but inhibits macrophage proliferation.

[0012] Furthermore, in the application provided by the present invention, galangin reduces the number of M1 macrophages.

[0013] Furthermore, in the application provided by the present invention, galangin increases the number of M2 macrophages.

[0014] Furthermore, in the application provided by the present invention, the drug comprises galangin and a pharmaceutically acceptable carrier.

[0015] In the present invention, the term "pharmaceutically acceptable carrier" refers to a substance that can be mixed with the active ingredient (i.e., galangin) in a pharmaceutical preparation, does not affect the efficacy and safety of the drug, and helps the preparation to form, store, transport, and exert its medicinal effect. These carriers can be reasonably selected according to the extract dosage form, drug properties, and clinical needs to ensure the safety and effectiveness of the drug.

[0016] According to the actual application needs, in the drugs provided by the present invention, according to the methods well-known to those skilled in the art, galangin can be prepared into a pharmaceutically acceptable salt of galangin as the active ingredient.

[0017] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of galangin and has no adverse effects biologically or otherwise. Pharmaceutically acceptable salts refer to converting the base group in the parent compound into a salt form, such as inorganic or organic acid salts of the base group (such as an amino group). Generally, the parent compound is reacted with conventional types of acids in a solvent system for preparation. Inorganic acids generally include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid, etc.; organic acids generally include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, or salicylic acid, etc.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0019] The present invention for the first time reveals that galangin effectively regulates the macrophage polarization balance by specifically binding to and targeting the degradation of ANXA2 protein in macrophages, reduces the number of pro-inflammatory M1 macrophages and increases the number of anti-inflammatory M2 macrophages, thereby inhibiting the inflammatory response and improving the pathological state of inflammatory skin diseases (such as psoriasis). Experimental data show that galangin selectively inhibits the proliferation of macrophages without affecting macrophage apoptosis, and its high affinity and specificity with ANXA2 are confirmed by SPR technology, molecular docking and mass spectrometry analysis, avoiding non-target binding with other members of the Annexin family or common proteins, indicating that galangin can be used as an excellent ANXA2-targeted drug and a drug for improving inflammatory skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is the induction curve of the binding energy between galangin and ANXA2.

[0022] Figure 2 It is the molecular docking diagram of galangin and ANXA2.

[0023] Figure 3Mass spectrometry analysis results of the binding of biotin-galangin to ANXA2, SLC25A5, and PKM, respectively. Among them, A is a schematic diagram of IP-MS detection; B is the galangin interacting proteins identified by mass spectrometry; C is the effect of galangin on ANXA2, SLC25A5, and PKM determined by CO-IP experiment.

[0024] Figure 4 Mass spectrometry analysis results of the binding of biotin-galangin to ANXA1, ANXA2, ANXA3, ANXA4, and ANXA5, respectively.

[0025] Figure 5 Test results of the improvement of skin conditions in mice with inflammatory skin disease models by galangin. Among them, A is a representative image of the back skin of mice; B is a representative image of H&E staining of skin sections of the back of mice.

[0026] Figure 6 Test results of galangin inhibiting macrophage proliferation. Among them, A is the cell viability detected by CCK-8 method after RAW 264.7 cells were treated with 2.5 μM, 5 μM, and 10 μM galangin for 24 hours; B is the representative pictures and statistical results of EdU staining; C is the apoptotic cells detected by AnnexinV-APC / PI staining combined with flow cytometry after RAW 264.7 cells were treated with 10 μM galangin for 24 hours; # indicates p < 0.05 compared with the control group.

[0027] Figure 7 Test results of the effect of galangin on M1 macrophages or M2 macrophages.

[0028] Among them, A is the expression levels of genes such as Cd40, IL-6, iNOS, and Cd68; B is the expression levels of genes such as Arg1, Mgl1, Mgl2, and YM1. Detailed implementation mode

[0029] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. %, in the following embodiments, is mass percentage unless otherwise specified. The ratios in the following embodiments are mass ratios unless otherwise specified.

[0030] Galangin was purchased from Jiangsu Yongjian Pharmaceutical Co., Ltd., and the product number is 103299.

[0031] Biotin-GA is biotin-labeled galangin, specifically, galangin is converted into an active ester and then reacted with the amino group of biotin to obtain biotin-labeled galangin. When preparing the biotin-GA used in the examples, the molar ratio of biotin to galangin is 1:1.

[0032] Example 1

[0033] This example provides a verification test for the targeted binding of galangin to ANXA2.

[0034] (1) Binding affinity of galangin to ANXA2.

[0035] The real-time interaction between ANXA2 and galangin was detected using Biacore T200 SPR technology. The ANXA2 protein was immobilized on the activated CM5 sensor chip by standard amine coupling method in PBS buffer (pH 7.4). Galangin was dissolved in DMSO. The binding of ANXA2 and galangin was carried out with running buffer (PBS containing 5% DMSO), and through serial dilutions from the stock solution, at a flow rate of 20 μL / min for 120 seconds. The kinetic dissociation constant (KD) was calculated by Biacore T200 evaluation software.

[0036] The results are as Figure 1 shown. After surface plasmon resonance analysis, galangin directly binds to the recombinant human ANXA2 protein with an affinity of KD = 6.74×10 -6 M.

[0037] (2) Molecular docking of galangin with ANXA2.

[0038] The crystal structure of the protein (ANXA2, PDB ID: 7pcb) was downloaded from the PDB database, and the three-dimensional structure of the small molecule was downloaded from the PUBCHEM database, and energy minimization was performed under the MMFF94 force field. The receptor protein was processed using PyMol 2.5.5, including removing water molecules, salt ions, and small molecules. Then, it was imported into Autodock Tools (version 1.1.2) to add hydrogen atoms, calculate the total charge, set the atom type, and save it in pdbqt format. Finally, the docking results were visualized and analyzed using PyMol 2.5.5.

[0039] The results are as Figure 2 shown. The docking score of galangin with ANXA2 is -6.526 kcal / mol.

[0040] (3) Specific targeted binding of galangin to ANXA2.

[0041] RAW 264.7 cells transfected with the specified plasmid were lysed with IP lysis buffer at 4 °C for 24 h. The samples were centrifuged at 12,000 g for 10 min and then incubated with free biotin or biotin-labeled galangin (biotin-GA) at 4 °C for 2 h. The beads were then washed approximately three times with PBS. The immune complexes were eluted in SDS loading buffer and subjected to immunoblot analysis as described above. For mass spectrometry (MS) analysis, the eluates were separated by 8–12% SDS-PAGE gels and stained. The bands were excised and the proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0042] The results are as Figure 3 shown, and potential GA-interacting proteins were identified by systematic mass spectrometry ( Figure 3 A). Previous studies have shown that PKM2, SLC25A5, and ANXA2 can interfere with cell proliferation and reduce the inflammatory response ( Figure 3 B). To confirm the interaction between GA and these proteins, biotin and biotin-GA were incubated with the cell lysates of HEK 293T cells. The results showed that only ANXA2 interacted with GA ( Figure 3 C).

[0043] HEK293T cells were transfected with HA-ANXA1, HA-ANXA2, HA-ANXA3, HA-ANXA4, or HA-ANXA5 plasmids in DMEM medium using PEI, and then the medium was replaced with DMEM medium containing 10% fetal bovine serum after 8 h. After 48 h, the collected cells were lysed in RIPA lysis buffer containing a protease and phosphatase inhibitor mixture. In vitro competitive pull-down assays were performed using recombinant ANXA2 protein to evaluate the direct interaction between GA and ANXA2.

[0044] The results are as Figure 4 shown, and the binding ability of galangin to other members of the annexin family was examined. The results showed that galangin was able to bind to ANXA2 and not to ANXA1, ANXA3, ANXA4, or ANXA5. These results indicate that GA selectively binds to ANXA2.

[0045] From the above results, it can be seen that GA can be used as a carrier for drug composition (such as biotin-GA), does not interact with other conventional proteins, and does not bind to other members of the annexin family, and has the function of specifically binding to ANXA2 target.

[0046] Example 2

[0047] This example provides an experiment on the improvement of skin conditions in mice with inflammatory skin disease models by galangin.

[0048] The hair on the tail and back of the mice in an area of 2 cm × 2 cm was shaved off, and 5% IMQ cream was applied to the tail and back continuously for 7 days, once a day, about 30 mg each time. The control group was given the same dose of petrolatum ointment. The mice in the drug administration group were given galangin cream (0.1 g / 10 g) every day in the modeling area.

[0049] Preparation of galangin cream: Weigh the excipients (see Table 1), put the required oil phase and water phase into different beakers, heat and melt them in a water bath at 85 °C, and homogenize; slowly pour the oil phase into the water phase, stir at a speed of 600 rpm for 5 min, and cool to room temperature to obtain an emulsion matrix. Galangin cream was prepared by the laboratory itself.

[0050] Table 1 Dosage of each excipient

[0051]

[0052]

[0053] The results are as Figure 5 shown. Compared with the control group, the IMQ-treated mice showed epidermal erythema on the 3rd day. The galangin-treated mice showed a reduction in skin erythema ( Figure 5 A). H&E staining showed that galangin significantly alleviated skin inflammation in IMQ-induced mice ( Figure 5 B).

[0054] Example 3

[0055] This example provides an experiment on the relationship between galangin and macrophages.

[0056] (1) Galangin inhibits macrophage proliferation.

[0057] RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and penicillin / streptomycin. RAW264.7 cells were placed in 96-well plates, synchronized under low-glucose culture conditions for 24 h, and then the high-glucose media with 0, 2.5, 5, and 10 μmol / L galangin were replaced respectively. After 24 h, CCK-8 was added and incubated for 2 h, and the optical density (OD) value was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated using the formula.

[0058] The results are as Figure 6 shown. Compared with the control group, the cells treated with galangin showed a significant decrease in the proliferation level ( Figure 6A). Subsequently, flow cytometry was used to evaluate the apoptotic changes of RAW 264.7 cells after galangin treatment ( Figure 6 B). The results showed that there was no significant difference in the proportion of apoptotic cells ( Figure 6 C).

[0059] (2) Galangin inhibits the proliferation of M1 macrophages and promotes the proliferation of M2 macrophages.

[0060] Total RNA was extracted from cells or skin tissues using an RNA extraction kit. Subsequently, reverse transcription (RT) was performed on 1 μg of purified total RNA using HiScript II Q RT SuperMix. 1 μg of purified total RNA was reacted with 4×gDNA wiper Mix at 42 °C for 2 minutes, and then with 5×HiScript III qRT SuperMix at 37 °C for 15 minutes and 85 °C for 5 seconds. qPCR amplification involved a premix of primers, cDNA samples, and SYBR qPCR SuperMix Plus, and quantitative PCR was performed using the QuantStudio TM 5 system. The relative expression levels of target genes were calculated using the 2-ΔΔCt method.

[0061] The results were as Figure 7 shown. The genes related to M1 macrophages (Cd40, Cd68, IL-6, and iNOS) in the skin tissues of IMQ-induced mice were significantly increased, while the genes of M2 macrophages (Mgll, Mgl2, Yml, and Argl) all decreased. Compared with that induced by IMQ, M1 macrophages decreased after galangin treatment ( Figure 7 A), and M2 macrophages increased ( Figure 7 B).

[0062] In summary, the present invention fully demonstrates that galangin specifically binds to ANXA2 and can target ANXA2 as a drug carrier to promote the degradation of ANXA2 in macrophages, thereby improving inflammatory skin diseases.

[0063] The above-described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents the preferred embodiments of the present invention. All other embodiments obtained by relevant deductions and substitutions made by those of ordinary skill in the art under the premise of not making creative efforts based on the concept of the present invention fall within the scope of protection of the present invention.

Claims

1. Use of galangin in the preparation of a drug targeting ANXA2, characterized in that, Galangin selectively binds to ANXA2.

2. The application according to claim 2, characterized in that, Galangin targets ANXA2 in macrophages.

3. Use of galangin in the preparation of a drug for degrading ANXA2, characterized in that, Galangin promotes the degradation of ANXA2.

4. The application according to claim 3, characterized in that, Galangin targets ANXA2 in macrophages, selectively binds to ANXA2, and promotes the degradation of ANXA2 in macrophages.

5. Application of galangin in the preparation of drugs for improving inflammatory skin diseases.

6. The application according to claim 5, wherein The inflammatory skin disease is psoriasis.

7. The application according to claim 5, characterized in that, Galangin does not affect the apoptosis of macrophages but inhibits macrophage proliferation.

8. The application according to claim 5, characterized in that Galangin reduces the number of M1 macrophages.

9. The application according to claim 5, wherein Galangin increases the number of M2 macrophages.

10. The application according to claim 5, wherein The drug includes galangin and a pharmaceutically acceptable carrier.

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