Application of 8-methyl quercetin in preparation of TLR7 immunomodulator

Abstract

The invention discloses an application of 8-methyl quercetin in preparation of a TLR7 (toll-like receptor 7) immunomodulator. According to the invention, it is found that 8-methyl quercetin can be used as a ligand of TLR7, and the 8-methyl quercetin has strong binding and interaction with swine TLR7 protein. Therefore, the 8-methyl quercetin can be used as an immunomodulator or a lead compound acting on the TLR7. As the TLR7 protein is conservative in evolution, the application of the 8-methyl quercetin is not limited to pigs, and the 8-methyl quercetin can also be applied to other species.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug development, and in particular relates to an application of 8-methylquercetin in the preparation of a TLR7 immunomodulator. Background Art

[0002] Toll-like receptor 7 (TLR7) is a crucial component of the innate immune response, playing a crucial role in resisting pathogen infection, anti-tumor immune responses, and cellular autophagy. TLR7 is primarily involved in the recognition of single-stranded RNA. Certain pathogen infections or tumors can negatively regulate the NF-κB signaling pathway, inhibiting the innate immune response and enabling immune escape. TLR7 agonists can promote the maturation or activation of immune cells, triggering adaptive immune responses, and enhancing the body's immune defenses against pathogens, with the potential for development as immunopotentiators. TLR7 inhibitors have the potential to alleviate inflammation and treat autoimmune diseases.

[0003] To date, researchers have designed and synthesized a variety of TLR7-targeting structures, but only a handful of these modulators have been successfully marketed. Furthermore, research on TLR7 modulators has focused on a few common core structures, lacking the exploration and modification of novel structures. Currently, there is limited research on 8-methylquercetin and flavonoids as TLR7 ligands. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a use of 8-methylquercetin in the preparation of a TLR7 immunomodulator.

[0005] The present invention is achieved by using 8-methylquercetin in the preparation of an animal TLR7 immunomodulator.

[0006] Preferably, the animals include humans and animals.

[0007] Preferably, the animals are livestock and poultry.

[0008] The present invention overcomes the shortcomings of the prior art and provides a method for using 8-methylquercetin as a TLR7 ligand in an immunomodulatory agent. Based on virtual screening, the present invention obtained a new compound, 8-methylquercetin, also known as 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-8-methyl-4H-chromen-4-one, which can serve as a TLR7 ligand. The structure is shown below.

[0009]

[0010] This study investigated the binding and interaction between 8-methylquercetin and porcine TLR7 protein and found that 8-methylquercetin can form a strong interaction with porcine TLR7 protein. Therefore, 8-methylquercetin can be used as an immunomodulator or lead compound acting on TLR7 ligands.

[0011] Compared to the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: 8-methylquercetin of the present invention can be used as a TLR7 ligand nucleus for the preparation of immunomodulators targeting TLR7, and can be applied to the prevention and treatment of livestock and poultry diseases. In addition, because the TLR7 protein is relatively conserved during evolution, the application of 8-methylquercetin of the present invention is not limited to pigs, but can also be applied to other species. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the interaction between 8-methylquercetin and porcine TLR7;

[0013] Figure 2 It is a molecular dynamics simulation analysis of 8-methylquercetin and Gardiquimod with porcine TLR7 protein;

[0014] Figure 3 The SPR method was used to determine the response of 8-methylquercetin to porcine TLR7 protein. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] 1. Molecular docking of 8-methylquercetin and porcine TLR7 protein

[0017] Discovery Studio (DS) software was used to process the receptor protein and ligand separately and perform molecular docking. The porcine TLR7 protein, as the receptor, was predicted using homology modeling on the Swiss-Model online platform (reference: CHAMIZO-GONZáLEZ F, GORDILLO B, HEREDIA F J. Elucidation of the 3D structure of grape seed 7S globulin and its interaction with malvidin 3-glucoside: A molecular modeling approach [J]. Food Chem, 2021, 347(129014).).

[0018] The receptor protein was processed using the Prepare protein module of the DS software to remove heteroatoms and water molecules, add hydrogen atoms, and supplement missing amino acid residues. Docking sites were set based on the binding sites of known TLR7 ligands. The small molecule ligand was processed using the Prepare Ligands module of the DS software to remove duplicate conformations, enumerate isomers and tautomers, and generate 3D conformations. After the receptor and ligand were processed, molecular docking was performed using CDocker in the Discovery Studio software. The processed protein and small molecule were set as receptors and ligands, respectively. The Cluster Poster value was set to 0.5, and all other parameters were left as default.

[0019] The results are as follows Figure 1 As shown in the figure, based on the molecular docking results, the molecular docking score (CDocker Energy) of 8-methylquercetin with porcine TLR7 protein was 51.21, which was higher than the score of the positive control compound Gardiquimod (17.6401). 8-Methylquercetin formed intermolecular interactions with TYR351*, GLN354*, and VAL355* (* indicates amino acid residues located on the B chain of the protein dimer) of porcine TLR7 through hydrogen bonds, formed a salt bridge with amino acid residue LYS462*, and formed interactions with ARG410*, ASP556, and THR587 through van der Waals forces. Residues such as TYR356*, PHE408*, ​​and ALA533 participated in hydrophobic interactions.

[0020] 2. Molecular dynamics simulation analysis of 8-methylquercetin and porcine TLR7 protein

[0021] Gromacs 2024.4 software was used to perform molecular dynamics simulations of two complexes of porcine TLR7 protein with 8-methylquercetin and Gardiquimod. Amber14sb was selected as the protein force field, Gaff2 was selected as the ligand force field, and the TIP4P water model was used to add solvent to the protein-ligand system and establish a water box with a periodic boundary of 1.2 nm. The particle-mesh mesh (PME) method was used to calculate long-range electrostatic interactions, and the Monte Carlo ion placement method was used to introduce an appropriate number of sodium and chloride ions to neutralize the charge of the entire system. Before the formal simulation, the system energy was minimized and balanced by performing the following three steps:

[0022] (1) Minimize the energy of each system using the steepest descent algorithm with 50,000 steps;

[0023] (2) Maintaining constant particle number, volume, and temperature (310 K), each system was pre-equilibrated for 50,000 steps with a step size of 2 fs;

[0024] (3) Maintaining constant particle number, pressure (1 atmosphere), and temperature (310 K), the system was pre-equilibrated for 50,000 steps with a step size of 2 fs. After the system energy was minimized and equilibrated, a molecular dynamics simulation was performed for 100 ns with a step size of 2 fs without any constraints, and the structural coordinates were saved every 10 ps.

[0025] Finally, the root mean square deviation (RMSD) in the molecular dynamics simulation trajectory and the structure comparison of the complex at five moments of 0, 25, 50, 75, and 100 ns were analyzed, and the average binding free energy between the protein and the ligand was calculated using the MM / GBSA method.

[0026] The RSMD curve is an indicator for judging the stability of the protein-ligand complex. The smaller the RMSD value, the more stable the complex. Figure 2 In A, the RMSD curve of the complex between 8-methylquercetin and porcine TLR7 protein is stable at From the perspective of RMSD, the complex formed with the positive control drug Gardiquimod and porcine TLR7 ( Figure 2 B) is equivalent, indicating that the complex formed by 8-methylquercetin and the target protein has good stability.

[0027] By comparing the conformations of the complex at five moments of molecular dynamics simulation, the binding stability of the complex in molecular dynamics simulation is studied. Figure 2 C, At five moments of 0, 25, 50, 75, and 100 ns, 8-methylquercetin was always bound to the active pocket of the target protein without significant changes, indicating that the compound formed a stable complex with the target protein. Figure 2 D is the complex formed by the positive control drug Gardiquimod and the target protein.

[0028] After the complex system was stabilized, the average binding free energy of 8-methylquercetin and Gardiquimod to porcine TLR7 was calculated using the MM / GBSA method. Figure 2 E, the average binding free energies of 8-methylquercetin and Gardiquimod to porcine TLR7 are -30.26 and -27.1 kcal / mol, respectively.

[0029] 3. SPR binding effect on porcine TLR7 protein

[0030] The present invention further verifies the response value of 8-methylquercetin to porcine TLR7 protein by surface plasmon resonance (SPR) test. Protein fixation was performed by amino coupling method. The compound was detected at a single concentration of 100 μM, and flowed at a speed of 10 μL / min for 150 s in each run. At the end of each flow, the chip was regenerated for 5 minutes with 10 mM glycine hydrochloride (pH 2.0) solution. The known TLR7 agonist Resiquimod was used as a positive control. Using Biacore Insight evaluation software, after subtracting the reference channel from the experimental channel, the data was globally fitted to the 1:1 Langmuir binding model to obtain the response unit (Resonance Units, RU).

[0031] The results are as follows Figure 3 As shown in the figure, the response value RU of 8-methylquercetin is 22.23, which is comparable to the positive reference (response value RU is 26.38), indicating that there is a strong binding between 8-methylquercetin and porcine TLR7 protein.

[0032] 4. Similarity analysis of TLR7 among different species

[0033] Sequence data for TLR7 from various species were downloaded from NCBI and analyzed for similarity across species. TLR7 is an evolutionarily conserved receptor with high similarity across species. The amino acid sequence similarity of porcine TLR7 protein with human, mouse, and rat TLR7 proteins was 83.33%, 77.62%, and 77.43%, respectively.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

Application of 1.8-methylquercetin in the preparation of TLR7 ligand or TLR7 immunomodulator for animals.

2. The use according to claim 1, characterized in that The animals include humans and animals.

3. The use according to claim 1, characterized in that The animals are livestock and poultry.

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