Application of 1, 2, 3, 4, 6-pentagalloylglucose in inhibition of peptidyl arginine deiminase

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of 1, 2, 3, 4, 6-pentagalloylglucose (PGG) in inhibition of peptidyl arginine deiminase (PAD4). The invention relates to an application of PGG in preparation of a medicine for inhibiting PAD4 in a subject or a biological sample. Experiments show that the moutan bark extract has a definite inhibiting effect on PAD4, and it is confirmed that moutan bark is a traditional Chinese medicine which is hopeful to screen out a natural PAD4 inhibitor. The PGG in the tree peony bark extract is confirmed to be a natural compound with a strong inhibition effect on PAD4 by analyzing bioactive components in the tree peony bark extract, and the PGG can provide support for developing a PDA4 inhibitor and preparing a medicine for treating PAD4-mediated diseases, obstacles or symptoms, and has very important significance.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of 1,2,3,4,6-pentagalloylglucose (PGG) in inhibiting peptidyl arginine deiminase (PAD4). Background Art

[0002] PAD4 catalyzes the post-translational modification of peptidyl arginine to peptidyl citrulline. Five PAD isozymes are known. PAD has diverse tissue distribution, different putative physiological functions, and is reported to be associated with a variety of disease states. Among them, PAD4 is responsible for the deimination or citrullination of a variety of proteins in vitro and in vivo, resulting in diverse functional responses in a variety of diseases. Typical diseases include rheumatoid arthritis, diseases in which neutrophils contribute to the pathogenesis (such as vasculitis, systemic lupus erythematosus, ulcerative colitis), and tumor indications. PAD4 inhibitors can be used as tools and therapeutic agents for human diseases through epigenetic mechanisms and have a wide range of applicability, such as PDA4 inhibitors can be used to combat rheumatoid arthritis, reduce pathological neutrophil activity in a variety of diseases, and treat cancer.

[0003] A variety of PDA4 inhibitors have been disclosed in the prior art, but there is no report on PGG as a PDA4 inhibitor.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to provide the use of PGG in inhibiting PAD4, which provides support for the development of PDA4 inhibitors and the preparation of drugs for treating PAD4-mediated diseases, disorders or symptoms, and is of great significance.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a use of PGG in preparing a medicament for inhibiting PAD4 in a subject or a biological sample.

[0008] The second object of the present invention is to provide the use of PGG in the preparation of a drug for treating a disease, disorder or condition mediated by PAD4.

[0009] More specifically, the PAD4-mediated disease, disorder or condition is selected from one or more of rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, tumors, acute lymphocytic leukemia and chronic acute lymphocytic leukemia.

[0010] The third object of the present invention is to provide a pharmaceutically acceptable composition, which comprises PGG.

[0011] Based on the aforementioned application of PGG, those skilled in the art can reasonably expect that a pharmaceutically acceptable composition containing PGG can be used to prepare a drug for inhibiting PAD4 in a subject or a biological sample, and can be used to prepare a drug for treating a PAD4-mediated disease, disorder or condition; of course, the PAD4-mediated disease, disorder or condition is selected from one or more of rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, tumors, acute lymphocytic leukemia and chronic acute lymphocytic leukemia.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention has found through experiments that the peony bark extract has a significant inhibitory effect on PAD4, and confirmed that the peony bark is a Chinese medicine with great potential for screening natural PAD4 inhibitors. Then, by analyzing the bioactive components in the peony bark extract, it is confirmed that PGG is a natural compound or lead compound in the peony bark extract that has a strong inhibitory effect on PAD4, which can provide support for the development of PDA4 inhibitors and the preparation of drugs for treating PAD4-mediated diseases, disorders or symptoms, and is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The SDS-PAGE Coomassie blue staining image of human His tagPAD4 expressed and purified in E. coli;

[0015] Figure 2 To verify the activity of His tag PAD4 using the classic COLDER method;

[0016] Figure 3 The dose inhibition curves of the known PAD4 inhibitors BB-Cl-amidine (A) and GSK484 (B);

[0017] Figure 4 The inhibitory effects of 12 traditional Chinese medicines on PAD4 at different concentrations;

[0018] Figure 5 The dose inhibition curves of water (A), 75% ethanol (B) and ethyl acetate (C) extracts of Paeonia suffruticosa L. on PAD4 inhibition;

[0019] Figure 6 Dose-inhibition curves of 75% ethanol extracts of Paeonia suffruticosa 90% from ten different batches of Paeonia suffruticosa 90% from the same origin for inhibiting PAD4 enzyme activity (AJ), showing the IC values ​​determined based on the ten curves50 Box plot of values ​​(K);

[0020] Figure 7 This is the dose inhibition curve of inhibiting PAD4 in different parts of peony;

[0021] Figure 8 The dose inhibition curves of white peony root (A) and red peony root (B) for inhibiting PAD4;

[0022] Fig. 9 Fingerprint analysis of Paeonia suffruticosa ethanol extract by HPLC-UV (A), PAD4 inhibition profile of LC fractions collected at 2-min intervals (B), and inhibition profile of selected fractions diluted 5-fold (C);

[0023] Fig.10 Base peak chromatogram of PGG, MS 1 Spectrum, MS 2 Spectra and peak areas;

[0024] Fig.11 The chemical structures of sixteen compounds identified from different parts of peony are shown;

[0025] Fig.12 The inhibitory effects of the sixteen compounds on PAD4 activity (AC) and the dose inhibition curve of PGG on PAD4 activity (D);

[0026] Fig.13 are the enzyme kinetic parameters of PGG to PAD4;

[0027] Fig.14 Molecular docking simulation of PGG and PAD4.

[0028] Description of main reference numerals:

[0029] exist Figure 3 In the figure, the inset diagram illustrates the chemical structure of the corresponding compound. Data are presented as mean ± SD (n = 3);

[0030] exist Figure 4 The inhibitory effects of water extracts of 12 traditional Chinese medicines on PAD4 at concentrations of 0.5 mg / mL (A), 1 mg / mL (B), and 2 mg / mL (C); the inhibitory effects of 75% ethanol extracts of 12 traditional Chinese medicines on PAD4 at concentrations of 0.5 mg / mL (A), 1 mg / mL (B), and 2 mg / mL (C); the inhibitory effects of ethyl acetate extracts of 12 traditional Chinese medicines on PAD4 at concentrations of 0.5 mg / mL (A), 1 mg / mL (B), and 2 mg / mL (C);

[0031] Figure 5In, data are presented as mean ± SD (n = 3);

[0032] Figure 6 In, data are presented as mean ± SD (n = 3);

[0033] Figure 7 In, curd (A), flower (B), leaf (C), pollen (D) and seed powder (E); in F, 1, curd; 2, leaf; 3, flower; 4, root; 5, pollen; Figure 7 The illustrations in the book are pictures of various parts of the peony;

[0034] Figure 8 In, data are presented as mean ± SD (n = 3);

[0035] Fig. 9 In, data are presented as mean ± SD (n = 3);

[0036] Fig.10 In the chromatogram of the base peak of pentagalloyl glucose (PGG), the retention time is 19.64min and the intensity is 2.51E8 (A); the MS of PGG 1 Spectrum, m / z is 939.1103 (z=1) and 469.0513 (z=2) (B); MS of PGG 2 Spectrum, m / z is 939.1103, the m / z of secondary fragment ions are 125.0243, 169.0141, 617.0784, 769.0891 (C); the peak area of ​​m / z 939.110 ((PGG)) in each fraction (D);

[0037] Fig.12 In, data are presented as mean ± SD (n = 3);

[0038] Fig.13 Data are expressed as mean ± SD (n = 3); Michaelis-Menten plot (A), Lineweaver-Burk plot (B) and the second plot of the slope in the Lineweaver-Burk plot (C) of PGG against PAD4; DARTS method was used to verify the interaction between purified PAD4 and PGG (D);

[0039] Fig.14 In the figure, the overall and local graphs of the molecular docking simulation (A) and the two-dimensional graph of the interaction between PGG and PAD4 amino acid residues (B). DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0041] 1. Experimental Materials and Methods

[0042] 1.1 Reagents and Chemicals

[0043] Chinese medicinal materials were purchased from Beijing Tong Ren Tang (Shanxi), α-N-benzoyl-L-arginine ethyl ester (BAEE), antipyrine, and 2,3-butanedione monoxime were purchased from Shanghai Yuan Ye Hua Technology Co., Ltd. (Shanghai, China), and L-citrulline, dithiothreitol (DDT), ammonium ferric sulfate dodecanobate, and phosphoric acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China);

[0044] Different batches of peony bark come from different limited liability companies;

[0045] Apigenin, apigenin, pentopyranose glucopyranose, methyl gallate, luteolin, paeoniflorin, isorhamnetin, ethyl gallate, apigenin 7-O-glucoside, osthole glycoside, astragaloside, paeoniflorin, phenoxypeoniflorin, and oxypeoniflorin were purchased from Chengdu Mante Biotechnology Co., Ltd. (Chengdu, China);

[0046] Quercetin and isorhamnetin 3-O-glucoside were purchased from Wuhan Tianzhi Biotechnology Co., Ltd. (Wuhan, China).

[0047] 1.2 Expression and purification of PAD4 enzyme

[0048] This study aims to find new PAD4 inhibitors from traditional Chinese medicine. Therefore, high-purity and high-activity PAD4 is required.

[0049] The pGEX vector containing the His-tag PAD4 sequence was transfected into E. coli BL21(DE3) and a similar expression and purification protocol was followed. Briefly, when the OD of the bacterial culture was 600When the value reached 0.6, isopropyl β-D-Thiogalactoside (IPTG) solution was added to a final concentration of 0.5 mM, and protein expression was induced at 16°C for 16 h. After centrifugation at 8000 rpm / min for 20 min, the precipitate was resuspended in PBS buffer (pH = 7.4) containing 10% glycerol and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The dissolved protein collected after centrifugation was loaded into a Ni-NTA column for binding. After binding, the resin was washed with PBS buffer containing 50 mM imidazole, and the protein was eluted with 250 mM imidazole. The eluted solution was further separated, and the purity was confirmed by SDS-PAGE and Coomassie blue staining. Finally, the purified PAD4 solution was stored at -80°C.

[0050] 1.3 PAD4 enzyme activity assay

[0051] PAD4 enzyme activity was determined using a colorimetric method (COLDER) with slight modifications. Briefly, the total volume of the incubation mixture was 100 μL, consisting of 100 mM Tris-HCl (pH = 7.6), 2 mM BAEE, 20 mM Ca 2+ and 4 μM PAD4, and incubated at 37°C for 90 min. Then 200 μL of freshly prepared color reagent (containing 0.5 mM antipyrine, 20 mM diacetyl monoxime, 1.5 mM ammonium iron (III) sulfate dodecahydrate, 2.2 M H3PO4, and 4.5 M H2SO4) was added to quench the reaction. The solution was then incubated at 98°C for 20 min to develop color, and then 200 μL of the reaction solution was transferred to a well plate and spectrophotometrically analyzed at a wavelength of 490 nm using a SuPerMax 3000FA microplate reader (Shanghai Shanpu Biotechnology Co., Ltd.).

[0052] 1.4PAD4 inhibition assay

[0053] The PAD4 inhibition test was performed using BAEE as a substrate, and the method was the same as above. At the same time, two known PAD4 inhibitors, GSK484 and BB-Cl-amidine, were used as positive controls. The dried Chinese medicinal materials were crushed with a grinder, and then the effective components of the Chinese medicine were extracted by reflux with water, 75% ethanol-water or ethyl acetate solution. The freeze-dried traditional Chinese medicine ethanol extract, ethyl acetate extract and water extract were dissolved in dimethyl sulfoxide and then diluted with water to a final concentration of 20 mg / mL, 10 mg / mL and 5 mg / mL. In the PAD4 inhibition experiment, 10 μL of traditional Chinese medicine extract was directly added to the PAD4 activity detection system, and 10 μL of water was used in the negative control experiment. Each experiment was carried out in three parallel reactions, and the absorbance was measured at a wavelength of 490 nm using an ELISA reader (SuperMax 3100, Shanghai Flash Spectrum Biotechnology Co., Ltd., Shanghai, China). The inhibition rate (Inhibition%) of traditional Chinese medicine extracts and compounds on PAD4 was calculated based on the comparison of the absorbance of citrulline derivatives in the presence or absence of inhibitors (control samples). The calculation formula is as follows:

[0054]

[0055] Among them, A inhibitor and A control is the absorbance of PAD4 with and without inhibitors / TCM extracts, and A inhibitorblank and A blank is the absorbance under blank conditions with and without the addition of inhibitors / TCM extracts.

[0056] 1.5 High performance liquid chromatography-ultraviolet (HPLC-UV) analysis

[0057] High-performance liquid chromatography fractionation of Paeonia suffruticosa was performed using an HPLC system equipped with a P-1100 pump and a D1100 UV detector (Dalian Yilite Analytical Instrument Co., Ltd., Dalian, China). The samples were separated using a VenusilXBP C18 column (4.6 mm × 150 mm, 5 μm) from Agilent Analytical Instrument Co., Ltd., and the detection wavelength was 280 nm. The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile in water (B), respectively, and the injection volume was 30 μL. The gradient separation flow rate was 1.0 mL / min, and the tailing information is shown in Table S1 below. The liquid fractions of Paeonia suffruticosa were continuously collected at intervals of 2 min from 0 to 32 min, and the total HPLC separation time for each sample was 32 min. All 16 liquid fractions were dried using a freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) and redissolved in H2O before use.

[0058] Table S1 HPLC gradient for separation of Paeonia suffruticosa extract

[0059]

[0060] 1.6 Ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry component identification

[0061] The main bioactive components in the Paeonia suffruticosa extract fractions were identified using a Q Exactive Plus mass spectrometer and a Dionex Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Germany). 18 The chromatographic column was (100×2.1 mm, 1.7 μm, Waters, USA), the flow rate was 100 μL / min, and the mobile phases were acetonitrile (A) and 0.1% formic acid aqueous solution (B).

[0062] The gradient elution conditions were set as follows: 0-1 min, 3% B; 1-4 min, 3%-5% B; 4-23 min, 5%-30% B; 23-26 min, 30%-95% B; 26-28 min, 95%-5% B; 28-30 min, 5%-95% B; 3-19 min, 5%-30% B. The injection volume was 10 μL, and the column temperature was 30°C.

[0063] The mass spectrometry data were collected in data-dependent acquisition (DDA) mode, and the mass spectrometry parameters were set as follows: ESI used negative and positive ion modes. The specific instrument parameters were as follows: ion spray voltage (KV) +3.8 / -3.5, capillary temperature +325℃ / -320℃, sheath gas +35 / -30, auxiliary gas +10 / -5. MS 1 and MS 2 The resolutions are 70000 and 17500 respectively. Scan modes include full MS and data-dependent MS 2 (Top5), a step-wise NCE fragmentation pattern with NCE values ​​of 20, 40, and 60 was used, and both the positive and negative MS scan ranges were 100–1000 m / z.

[0064] 1.7 Drug affinity reaction target stability test

[0065] Purified PAD4 was dissolved in PBS buffer (pH = 7.4) containing 2% glycerol at a concentration of 0.4 mg / mL, and the PGG mother solution was prepared with 50% DMSO at a concentration of 5 mM. In the DARTS experiment, 80 μL PAD4 solution was mixed with 10 μL water and 10 μL PGG mother solution, and a gradient concentration gradient was performed. The optimized final concentration series of PGG was 0 μM, 1 μM, 5 μM, 10 μM and 50 μM. The total volume of DARTS was kept at 100 μL. Then, 60 ng proteinase K (0.006 mg / mL, 10 μL) (proteinase K: protein = 1: 800, mass ratio) was added to each sample except the positive group, and the original protein was digested at 25 ° C for 5 min, with a total volume of 110 μL. Then 27.5 μL SDS loading buffer was added to the sample and incubated at 98 ° C for 10 min. Finally, Coomassie brilliant blue staining was used to observe the protection band.

[0066] 1.8 Molecular docking

[0067] The protein crystal structures of key targets were collected from the PDB database (PDBID: 3APM), and the SDF files of the main active ingredients of the core drugs were obtained from the PubChem database. The Pymol-2.1.0 software was used to remove water molecules and small molecule ligands to optimize the targets. The proteins were hydrogenated and charged using AutoDock Tools-1.5.6 and saved in pdbqt format. The energy minimization of the compounds was performed using the PyRx built-in tool (OpenBabel). With the protein as the receptor and the compound as the ligand, the molecular docking was performed using vina-2.0 in the PyRx software, the binding energy was calculated, and the result file was output. Finally, the PyMol software was used to visualize the results. Its affinity (kcal / mol) value represents the binding ability of the two. The lower the binding ability, the more stable the binding between the ligand and the receptor. Discovery Studio 2020Client (https: / / discover.3ds.com / discovery-studio-visualizer-download) was used for visualization analysis (the lower the binding energy, the better the binding).

[0068] 1.9 Statistical analysis

[0069] All experiments were performed in triplicate and the data are presented as mean ± SD. 50Values ​​are based on dose inhibition curves, Log(inhibitor) versus response - variable slope (four parameters), and calculated using growth / sincosine, logarithmic functions in the OriginPro 2021 software package, with upper and lower asymptotes fixed at 120% and 0%, respectively. Primary screening and Ki values ​​were determined by nonlinear regression using GraphPad Prism 9.5.1 (GraphPad Software, Inc., La Jolla, USA).

[0070] 2. Results and Analysis

[0071] 2.1 Discovery of traditional Chinese medicine with PAD4 inhibitory activity

[0072] Recombinant human PAD4 with a His tag (PAD4) was purified using a Ni-NTA column. The SDS-PAGE images of the crude lysate supernatant and purified PAD4 are shown in Figure 1 The results showed that a highly purified PAD4 enzyme was prepared and its activity was verified by the classic COLDER method ( Figure 2 ). Then, the developed PAD4 activity detection method was validated using the known PAD4 inhibitors BB-Cl-amidine and GSK484. Figure 3 As shown in A, the IC of BB-Cl-amidine 50 The value was 1.49 μM, which is consistent with previous reports. In addition, the IC 50 The value was determined to be 0.74 μM, which is also consistent with previous studies. The above results clearly show that our PAD4 detection method is successful and can be used to screen natural inhibitors of PAD4.

[0073] Traditional Chinese medicine is a valuable source for the identification of natural enzyme inhibitors. In this study, we collected 12 Chinese herbal medicines that play an important role in the treatment of blood diseases. The chemical components of these 12 Chinese herbs were extracted using three different extraction methods, resulting in 36 different extracts. The inhibition rate of these Chinese herbal extracts was determined using the classic freezing method. The inhibition rate against PAD4 was evaluated at final concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, and the results are shown in Figure 4 and Tables S2-S4. Among all herbal products, Paeonia suffruticosa, Rubia cordifolia, and Folium Isatidis had strong PAD4 inhibitory activity ( Figure 4 ). The ethanol extract of Paeonia suffruticosa had the best inhibitory effect, and its water extract and ethyl acetate extract also showed high inhibitory efficacy. In contrast, the inhibition rate of other tested Chinese medicines on PAD4 activity was relatively weak even at high doses. In order to further verify the inhibitory effect of Paeonia suffruticosa extract on PAD4, we plotted the dose-inhibition curves of its three extracts. Figure 5As shown in Figure 2, the ethyl acetate extract and ethanol extract of Paeonia suffruticosa showed higher inhibitory effects, IC 50 The values ​​were 25.1 μg / mL and 53.8 μg / mL, respectively. In addition, although the inhibitory effect of the water extract was weaker, the IC 50 The above results clearly show that some compounds in Paeonia suffruticosa have high PAD4 inhibitory potency.

[0074] Table S2 Inhibition rate of 12 Chinese herbal medicine water extracts on PAD4 activity measured at different concentrations (%)

[0075]

[0076]

[0077] Table S3 Inhibition rate of PAD4 activity of 12 Chinese herbal medicine ethanol extracts at different concentrations (%)

[0078]

[0079] Table S4 Inhibition rate of PAD4 activity of ethyl acetate extracts of 12 traditional Chinese medicines at different concentrations (%)

[0080]

[0081] 2.2 Confirmation of the inhibitory effect of Paeonia suffruticosa

[0082] To further confirm the high inhibitory effect of Paeonia suffruticosa on PAD4 activity, we purchased 10 different batches of Paeonia suffruticosa from different manufacturers. Detailed product information is shown in Table S5. Since the solubility range of ethanol is wider than that of ethyl acetate, the preparation of ethanol extracts is simpler, and ethanol extracts also have a good inhibitory effect on PAD4. Therefore, we used 75% ethanol to extract the natural compounds in ten batches of Paeonia suffruticosa. Figure 6 As shown in AJ, we determined and plotted dose-inhibition curves for these extracts. 50 The values ​​ranged from 48.8 μg / mL to 134.9 μg / mL, with a median of 81.3 μg / mL ( Figure 6 K). These results clearly indicate that the results are very reliable and Paeonia suffruticosa is a promising traditional Chinese medicine for screening natural PAD4 inhibitors.

[0083] Peony bark is the root bark of the peony family. Next, we tested the inhibitory effects of other parts of the peony family on the PAD4 enzyme. We collected the flower balls, flowers, leaves, pollen and seed cakes of the peony and prepared their ethanol extracts using the same method. The dose inhibition curves of these five extracts on PAD4 are shown in Figure 7 AE, for which the diagram is shown in Figure 7 F. Among the five extracts, the seed meal was found to have the strongest inhibitory activity against PAD4, with IC 50 The value was 36.0 μg / mL, followed by curd (44.8 μg / mL) and leaves (145.8 μg / mL).

[0084] According to systematic botany, peony belongs to the Paeoniaceae family, which also includes paeonia lactiflora. In traditional Chinese medicine, there are two products related to peony, namely white peony and red peony. The inhibition rate of ethanol extracts of white peony and red peony on PAD4 activity was then studied. Figure 8 As shown, dose inhibition curves were plotted and IC 50 The values ​​were 95.9μg / mL and 361.1μg / mL, respectively. The inhibitory effect of peony on PAD4 was similar to that of tree peony, indicating that there are highly potential natural PAD4 inhibitors in Paeoniaceae plants.

[0085] Table S5 Information of different batches of Paeonia suffruticosa

[0086]

[0087]

[0088] 2.3 Identification of bioactive components in Paeonia suffruticosa extract

[0089] Traditional Chinese medicine extracts are complex mixtures of multiple compounds. Next, a practical strategy based on bioactivity was adopted to identify the bioactive components in Paeonia suffruticosa extracts by chemical fingerprint analysis combined with in vitro PAD4 activity detection. Fig. 9 As shown in A, the extract of Paeonia suffruticosa was separated by HPLC-UV. The fractions were collected at intervals of 2 min and then freeze-dried. Each fraction was dissolved and used for PAD4 inhibition test. The results showed that the inhibition rates of F6, F7, F8 and F9 fractions were all higher than 50%, among which the inhibition rate of F8 fraction was the highest (>90%) ( Fig. 9 B). In order to further explore the inhibitory effects of the above four fractions, the fractions were diluted 5 times and then subjected to PAD4 inhibition experiments. The results showed that the inhibition rate of the F8 fraction was still the highest ( Fig. 9 C). According to the above results, the above four fractions, especially the F8 fraction, are biologically active fractions. Therefore, these four fractions were further characterized by LC-MS / MS to identify natural compounds with high PAD4 inhibitory potency.

[0090] 2.4 Identification of natural compounds in Paeonia suffruticosa extract

[0091] To further identify the main components with high inhibitory effects on PAD4, LC-MS / MS identification was performed on fractions F6, F7, F8, and F9. With the help of standards and previously reported literature research, a total of five compounds were identified, and the detailed information of their m / z fragment ions is shown in Table S6. Among these compounds, PGG had the largest peak area in fraction F8, with a retention time of 19.64, [MH] - The m / z of is 939.1103, [M-2H] 2- The m / z is 469.0513 ( Fig.10 ).like Fig.10 C, MS 1 and MS 2 The spectra all confirmed the identification of PGG and PGG was identified in the above four fractions. In addition, among the peak areas of m / z 939.1103 extracted, the peak area of ​​F8 was the largest, indicating that PGG was mainly located in the F8 fraction.

[0092]

[0093]

[0094] Table S7 Preliminary screening of the inhibitory rates of 16 compounds from tree peony on PAD4

[0095]

[0096]

[0097] 2.5 Kinetic analysis of the inhibition of PAD4 by PGG in Paeonia suffruticosa

[0098] In order to evaluate the inhibitory mechanism of PGG on PAD4, inhibition kinetic analysis was performed. According to the Michaelis-Menten equation, the inhibition kinetic behavior of different concentrations of PGG and substrate was studied. In the absence of PGG, the Km and Vmax values ​​were 2.83 mM and 8.78 μM / min / g protein, respectively. Fig.13 AB). In the presence of PGG, the Km value was significantly reduced, and the corresponding Vmax also gradually decreased, indicating that PGG inhibited PAD4 activity in a mixed inhibition manner, including non-competitive and non-competitive modes ( Fig.13 C).

[0099] To further confirm the direct interaction between PGG and PAD4, a DARTS experiment was performed. In the DARTS method, proteinase K was added to a protein solution containing PAD4 and without PGG. If PAD4 binds to PGG, its conformation may change, which will affect its ability to resist cleavage by proteinase K. Fig.13As shown in D, the addition of PGG increased the ability of PAD4 to resist proteinase K digestion, indicating that PAD4 is a direct target of PGG.

[0100] 2.6 Molecular docking

[0101] Molecular docking was performed to study the interaction mode and action site of PGG and PAD4. Fig.14 As shown in A, the average binding energy of PGG is -9.6 kcal / mol, indicating that PGG has a strong binding ability with PAD4.

[0102] The docking simulation results showed that PGG can form conventional hydrogen bonds with Gln349, Thr647, Leu410, Glu411, His471, and Asn301, and with Gly408, Asn588, Pro584, Pi-Anion and Leu140, Alkyl and Cys645, and Pi-Alkyll and Pro599. These observations suggest that PGG may have strong hydrogen bond interactions with PAD4 and partially explain the binding mode and site of PGG-PAD4 interaction ( Fig.14 B) These observations suggest that PGG may have strong hydrogen bonding interactions with PAD4 and partially explain the binding mode of PGG to PAD4.

[0103] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. Use of 1,2,3,4,6-pentagalloylglucose (PGG) in the preparation of a drug for inhibiting peptidyl arginine deiminase (PAD4) in a subject or a biological sample.

2. Use of PGG in the preparation of drugs for treating PAD4-mediated diseases, disorders or conditions.

3. The use according to claim 2, characterized in that: The PAD4-mediated disease, disorder or condition is selected from one or more of rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, tumors, acute lymphocytic leukemia and chronic acute lymphocytic leukemia.

4. A pharmaceutically acceptable composition, characterized in that The pharmaceutically acceptable composition includes PGG.

5. Use of the pharmaceutically acceptable composition according to claim 4 in the preparation of a medicament for inhibiting PAD4 in a subject or a biological sample.

6. Use of the pharmaceutically acceptable composition according to claim 4 in the preparation of a medicament for treating a disease, disorder or condition mediated by PAD4.

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