Use of epigallocatechin gallate in the preparation of a baza virus helicase inhibitor or an anti-baza virus drug

Abstract

This invention provides the application of epigallocatechin gallate (EGCG) in the preparation of Bagaza virus helicase inhibitors or anti-Bagaza virus drugs, belonging to the field of pharmaceutical technology. Utilizing the crystal structure of BAGV helicase, molecular docking analysis shows that EGCG has a dual inhibitory effect on both the RNA binding site and NTP binding site of BAGV helicase, thereby precisely inhibiting helicase activity. Furthermore, ATPase activity testing results demonstrate that EGCG has a significant inhibitory effect on BAGV helicase, thus achieving the goal of anti-BAGV. This invention provides a feasible strategy for innovative BAGV therapies.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of epigallocatechin gallate in the preparation of Bagaza virus helicase inhibitors or anti-Bagaza virus drugs. Background Technology

[0002] Bagaza virus (BAGV) is a mosquito-borne virus that can spread to other regions and even continents through bird migration. Evidence suggests that BAGV can cross the blood-brain barrier in birds and invade the brain, leading to death. Furthermore, autopsies of dead birds show severe damage to the heart, brain, kidneys, and other vital organs, posing a significant threat to vertebrate life. Notably, some patients with encephalitis have tested positive for anti-BAGV antibodies, indicating that BAGV can infect humans and cause encephalitis. Currently, research on BAGV is limited, and there are no targeted antiviral treatments or effective vaccines developed. To prevent this virus from becoming a threat to human health and safety, research into the treatment and prevention of BAGV is imperative.

[0003] Like other flaviviruses, the BAGV genome is translated into 10 proteins, including 3 structural proteins and 7 non-structural proteins. The non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, are involved in BAGV viral replication. The C-terminus of NS3 acts as a helicase in BAGV replication, catalyzing the unwinding of double-stranded RNA by providing energy through ATP hydrolysis. Besides catalyzing RNA unwinding, helicases play multiple important roles in the viral life cycle, including ribosome biogenesis, RNA splicing, export, maturation, and translation. Therefore, helicases could serve as key targets for anti-BAGV drug discovery.

[0004] Discovering new drugs is a time-consuming process, requiring substantial budgets and often with a low success rate. Plants and their derivatives provide an important and valuable reservoir for the exploration and development of novel antiviral drugs, as they are abundant in the natural environment and are expected to have few side effects. Epigallocatechin-3-gallate (EGCG), a monomeric catechin extracted from green tea, is a major bioactive component of tea polyphenols. Its diverse pharmacological properties include antibacterial, antiviral, antioxidant, anti-atherosclerotic, antithrombotic, anti-angiogenic, anti-inflammatory, and antitumor effects. EGCG can be used as a standalone therapeutic agent or in combination with other compounds, and its potential utility in diseases including cancer, metabolic disorders, neurodegenerative diseases, and microbial infections has been extensively studied. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of epigallocatechin gallate in the preparation of BAGV helicase inhibitors or anti-BAGV drugs, wherein the EGCG can significantly inhibit the helicase activity of BAGV, thereby blocking BAGV replication and achieving an anti-BAGV effect.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of epigallocatechin gallate in the preparation of Bagaza virus helicase inhibitors.

[0008] Preferably, the epigallocatechin gallate inhibits the ATPase activity of the Bagaza virus helicase.

[0009] Preferably, the effective concentration of the epigallocatechin gallate is not less than 6 μM.

[0010] Preferably, the structural formula of the epigallocatechin gallate is:

[0011]

[0012] This invention also provides the application of epigallocatechin gallate in the preparation of anti-Bagaza virus drugs.

[0013] Preferably, the effective concentration of the epigallocatechin gallate is not less than 6 μM.

[0014] Preferably, the structural formula of the epigallocatechin gallate is:

[0015]

[0016] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0017] Preferably, the drug dosage form includes tablets, pills, granules, solutions, injections, or capsules.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides the application of epigallocatechin gallate (EGCG) in the preparation of Bagaza virus helicase inhibitors or anti-Bagaza virus drugs. Utilizing the crystal structure of BAGV helicase, molecular docking analysis demonstrates that EGCG has a dual inhibitory effect on both the RNA binding site and NTP binding site of BAGV helicase, thereby precisely inhibiting helicase activity. Furthermore, ATPase activity testing results show that EGCG has a significant inhibitory effect on BAGV helicase, thus achieving the goal of anti-BAGV. This invention provides a feasible strategy for innovative BAGV therapies. Attached Figure Description

[0020] Figure 1 The image shows the results of BAGV helicase purification.

[0021] Figure 2 Image of stick-shaped protein crystals obtained from BAGV helicase protein crystal culture;

[0022] Figure 3 The results show the molecular docking of EGCG with BAGV helicase;

[0023] Figure 4 In diagram A, EGCG inhibits the ATPase activity of BAGV helicase; in diagram B, a double reciprocal plot fitted according to the Michaelis-Menten equation is shown. Detailed Implementation

[0024] This invention provides the application of epigallocatechin gallate in the preparation of Bagaza virus helicase inhibitors.

[0025] In this invention, the BAGV helicase gene sequence was synthesized and molecularly cloned, followed by induced expression, large-scale bacterial culture, and purification of the target protein helicase. The homogeneous protein with a purity exceeding 95% was then crystallized, and high-quality crystals were harvested and subjected to X-ray diffraction at the Shanghai Synchrotron Radiation Facility. The obtained diffraction data were analyzed, and analysis of the BAGV helicase crystal structure revealed that helicase possesses an important substrate-binding pocket, which can be used to design antiviral drugs. Finally, high-resolution X-ray diffraction was obtained. The 3D protein structure was obtained. Molecular docking of BAGV helicase with EGCG revealed favorable binding parameters at both the BAGV helicase NTP and RNA sites. This invention also reflects changes in BAGV helicase activity by testing ATPase activity, measuring ATPase activity with and without EGCG. The results show that EGCG indeed has a significant inhibitory effect on BAGV helicase activity.

[0026] In this invention, the epigallocatechin gallate inhibits the ATPase activity of the Bagaza virus helicase. The effective concentration of the epigallocatechin gallate is preferably not less than 6 μM. The effective concentration refers to the concentration at which the therapeutic agent treats, alleviates, or prevents the target disease or virus. The structural formula of the epigallocatechin gallate is:

[0027]

[0028] This invention also provides the application of epigallocatechin gallate in the preparation of anti-Bagaza virus drugs.

[0029] In this invention, during the preparation of the Bagaza virus drug described above, the effective concentration of the epigallocatechin gallate is preferably not less than 6 μM. The effective concentration refers to the concentration at which the therapeutic agent treats, alleviates, or prevents the target disease or virus. The structural formula of the epigallocatechin gallate is:

[0030]

[0031] The preferred pharmaceutical formulation also includes pharmaceutically acceptable excipients, such as one or more of diluents, colorants, sweeteners, coating agents, binders, absorbents, disintegrants, releasing agents, dispersants, humectants, solubilizers, buffers, and surfactants. The preferred dosage form includes tablets, pills, granules, solutions, injections, or capsules. The pharmaceutical formulation described in this invention can be administered orally, intraperitoneally, subcutaneously, intramuscularly, intravenously, or nasally.

[0032] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In the following examples, EGCG was purchased from Sigma-Aldrich, product number E4143.

[0035] Example 1

[0036] Effect of epigallocatechin gallate (EGCG) on BAGV helicase protein

[0037] 1. The crystal structure of BAGV Helicase protein was determined using structural biology methods such as molecular cloning, protein expression and purification.

[0038] 1.1 Molecular Cloning

[0039] The full-length BAGV helicase gene (GenBank: AWA45345.1) was downloaded from NCBI and synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on the codon preference of E. coli. Suitable forward and reverse primers were designed to introduce restriction endonucleases BamHI and XhoI. The forward primer BAGV-BamHI-F: CGGGATCCATGCTGCGTAAACGTCA (SEQ ID No. 1) and the reverse primer BAGV-XhoI-R: CCGCTCGAGTTAACGTTTACCGCAC (SEQ ID No. 2) were used for molecular cloning. The target fragment was ligated into the target vector pET.32M.3C. After sequencing confirmed that there were no mutations, the target plasmid was expressed in E. coli DE21 (DE3) cells (Solarbio, China).

[0040] The nucleotide sequence of the BAGV helicase is as follows:

[0041]

[0042] The amino acid sequence of BAGV helicase is as follows:

[0043] MLRKRQLTVLDLHPGSGKTRKVLPQIVKTAIDRRLRTAILAPTRVVAAEIAEALKEYPIRYLTPAVKREHTGTEIIDVMCHATLTSRLLTPQRVPNYNLFVMDEAHFTDP ASIAARGYISTKVELGEAAAIFMTATPPGTRDPFPDSNSPIVDVEEQIPDRAWNSGYEWITDYTGKTVWFVPSVKMGNEIAVCLTKAGKKVIQLNRKSFDSEYPKCKTGE WDFVITTDISEMGANFGASRVIDSRKCIKPVIIEDGEGSVQLNGPVPITAASAAQRRGRIGRSYVQVGDEYHFSGPTSEDDHDFAHWKEAKILLDNINLPNGLVAQLYEP EREKVFSIDGEYRLRTEQRKNFVEFLRTGDLPVWLSYKLAEAGVAYHDRKWCFDGPSINTVLEDNNPVELWTKSGEKKILRPRWRDGRLWADHQALKSFKDFACGKR(SEQ IDNo.4).

[0044] 1.2 Protein Expression

[0045] E. coli containing the target protein were first cultured in 5 mL LB medium containing 100 μg / mL ampicillin at 37°C overnight. The next day, they were transferred to 1 L of liquid medium and cultured at 37°C for 4 h. Then, 1 mM isopropyl-β-d-thiogalactoside (IPTG) was added and the culture was incubated overnight at 16°C to induce protein expression. Finally, the bacterial cells were centrifuged in batches at 4000 rpm for 25 min. The supernatant was discarded, and the bacterial pellet was resuspended in buffer A (20 mM Na2HPO4, pH 8.0, 0.5 M NaCl, 20 mM imidazole).

[0046] 1.3 Protein purification

[0047] E. coli containing the target protein were sequentially lysed using high-pressure homogenization and ultrasonic cell disruption. The lysate was centrifuged at 4°C, 15000 rpm for 1 h in a refrigerated centrifuge. The supernatant was collected, incubated with Ni medium, and then purified by affinity chromatography. The target protein was obtained by elution with buffer B (20 mM Na₂HPO₄, pH 8.0, 0.5 M NaCl, 250 mM imidazole) and digested with PreScission protease overnight at 4°C. Ion exchange chromatography was then performed after digestion. The tubing and column of the AKTA purification instrument were equilibrated beforehand with buffer C (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), pH 6.8, 50 mM NaCl, 5% glycerol) and buffer D (50 mM HEPES, pH 6.8, 1 M NaCl, 5% glycerol). The protein-containing solution from the previous step was desalted to below 100 mM NaCl and concentrated to 5 mL. The solution was then purified by HiTrap. TM SP HP uses a NaCl concentration gradient to elute the target protein, and the target protein is finally eluted at a NaCl concentration of 250 mM.

[0048] Next, gel filtration chromatography was performed to concentrate the target protein collected in the previous step to 500 μL, which was then injected into Superdex. TM Use a 200Increase 10 / 300GL column to elute the target protein with buffer E (10mM Tris-HCl, pH 8.0, 150mM NaCl, 5mM MTT, 5% glycerol).

[0049] Samples were retained and analyzed by SDS-PAGE electrophoresis at each step above. Figure 1 The results showed that BAGV helicase protein with high purity and good homogeneity was obtained, with a purity of over 95%. The concentration was adjusted to 7.5 mg / mL and 15 mg / mL using 30 kDa concentration tubes.

[0050] 1.4 Crystal Culture

[0051] 15 mg / mL BAGV helicase protein was grown in a 16°C crystallization incubator via a seated-drop vapor diffusion method. Stick-shaped protein crystals were finally obtained in the pool solution (0.2 M sodium malonate hydrate, pH 6.0, 20% w / v polyethylene glycol 3350) (see [link to crystallization incubator]). Figure 2 These crystals were preserved by liquid nitrogen flash freezing in a 20% glycerol antifreeze solution and transported to the Shanghai Synchrotron Radiation Facility to obtain diffraction data. The results are shown in Table 1.

[0052] Table 1. Collection and detailed statistics of BAGV helicase diffraction data

[0053]

[0054]

[0055] a The value in parentheses represents statistics for the highest resolution shell.

[0056] b Rmerge=ΣhklΣj|Ihkl-Ihkl(J)| / ΣhklΣj|Ihkl(J)|.Ihkl(J)and Ihkl means

[0057] JTH and average reflectance hkl.

[0058] c Rwork = Σhkl||Fobs|-|Fcalc|| / |Fobs|. Fobs and Fcalc represent the observed and calculated structure factors, respectively. Rfree is the R-factor calculated using 10% unique reflectance as the test set.

[0059] d This indicates that these values ​​are reported by PHENIX.

[0060] The results in Table 1 indicate that the diffraction data show that the crystal is BAGV helicase protein.

[0061] 2. The BAGV helicase was molecularly docked with the inhibitor EGCG to analyze their interaction mode, determine the interaction between the inhibitor EGCG and protein residues, and found that the RNA / ATP site of the helicase binds well to the inhibitor EGCG.

[0062] 2.1 Preparation of EGCG molecular structure

[0063] The EGCG molecular structure used in this docking was obtained from the PubChem database and transformed and minimized using Chem3D. All structures were then imported. The software establishes a database of ligand molecules through hydrogenation, structure optimization, and energy minimization for molecular docking.

[0064] 2.2 Preparation of helicase protein structure

[0065] The obtained diffraction data were processed using Maestro 11.9 software to analyze the structure of the BAGV helicase protein. The protein preparation wizard in the software was used to remove water of crystallization, add missing hydrogen atoms, repair missing bond information, repair missing peptides, and finally perform energy minimization and geometric optimization.

[0066] 2.3 Molecular docking

[0067] use Molecular docking was performed using the Glide module in Maestro software. Protein preparation was performed using the protein preparation wizard module. Receptors were pretreated, optimized, and minimized. All molecules were prepared using the default settings of the LigPrep module. During screening in the Glide module, the prepared receptors were imported, and appropriate locations were specified in the receptor grid generation. Predicted protein binding sites were selected. The center of the frame. Finally, molecular docking and screening are performed using the standard precision (SP) method.

[0068] 2.4 Screening and Analysis of Dating Results

[0069] The interaction mechanism between compound EGCG and the target protein helicase was analyzed to determine the interactions between EGCG and protein residues, such as hydrogen bonds, π-π interactions, and hydrophobic interactions. The docking fraction of the compounds was also considered to infer whether the screened compounds possessed certain activity. The molecular docking results are shown in Table 2.

[0070] Figure 3 The results in Table 2 indicate that EGCG has a significant interaction with the RNA binding site of the helicase, and EGCG also has a strong binding affinity for the NTPase site of BAGV helicase. In other words, EGCG has a dual inhibitory effect on both the RNA binding site and the NTP binding site of BAGV helicase, thereby precisely inhibiting the activity of BAGV helicase.

[0071] Table 2 Molecular docking results.

[0072]

[0073] 3. Perform enzyme activity assays on the inhibitor EGCG, and compare the data with normal enzyme activity to determine whether the inhibitor EGCG can inhibit the ATPase activity of BAGV helicase, thus judging the inhibitory effect.

[0074] 3.1 Assay of ATPase activity

[0075] The absorbance was measured at 630 nm using a SpectraMax iD3 multifunction reader, following the instructions for the Malachite Green Phosphate Detection Kit (Beyotime, Shanghai, China). K was calculated based on the measured OD value and the standard curve. m and K cat Using GraphPadPrism software, a double reciprocal plot was generated to compare the levels of ATPase activity and calculate K. i .

[0076] The principle of ATPase activity detection is that malachite green, molybdate, and free inorganic orthophosphate form a green phosphomolybdic acid complex. The absorbance of this green phosphomolybdic acid complex at 620-640 nm is proportional to the concentration of free inorganic phosphate in the solution. BAGV helicase hydrolyzes ATP to release free inorganic phosphate. After the green phosphomolybdic acid complex is added to the kit, the rate of inorganic phosphate formation can be calculated by using a standard curve and absorbance.

[0077] ATPase activity assay: ATP activity inhibition was performed using 313 nM BAGV helicase protein and 6 μM EGCG (Sigma-Aldrich, Darmstadt, Germany). Three ATPase activity inhibition assays were conducted in 96-well plates. 25 μL of 0 or 6 μM EGCG and 40 μL of 313 nM BAGV helicase protein were added and reacted for 5 min, followed by the addition of 65 μL of different concentrations of ATP. The mixture was incubated at 25°C for 45 min. The reaction was terminated by adding 70 μL of reagent. After incubation at room temperature for 30 min, the absorbance was measured at 630 nm using a SpectraMaxiD3 multifunction detector.

[0078] Depend on Figure 4 The results showed that the rate of free inorganic phosphate formation after the addition of EGCG was significantly lower than that without the addition of EGCG, indicating that EGCG effectively inhibited the helicase activity of BAGV.

[0079] K was calculated after plotting using Graphpad Prism. i =0.599±0.390μM.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of epigallocatechin gallate in the preparation of anti-Bagaza virus drugs.

2. Use according to claim 1, characterized in that, The effective concentration of the epigallocatechin gallate is not less than 6 μM.

3. Use according to claim 1 or 2, characterized in that, The structural formula of the epigallocatechin gallate is: 。 4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, pills, granules, solutions, injections, or capsules.

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