A model of the binding of evodiamine with AMPK protein and a method for constructing the model

Abstract

The application discloses a combination model of evonik secondary alkaline and AMPK protein and a construction method thereof, and belongs to the technical field of computer-aided drug design and structural biology. The application firstly constructs an evonik secondary alkaline AMPK precise combination model verified by molecular docking and 100 ns all-atom molecular dynamics, and determines that the evonik secondary alkaline is combined with an AMPK active pocket, forms a key hydrogen bond with Val98, and forms a stable hydrophobic network with Ala45, Lys47, Tyr97 and Leu148; the binding free energy is ‑10.0 kcal / mol, the MM / PBSA binding free energy is ‑35.73 kcal / mol, the conformation is stable, the binding is firm, and the thermodynamics is favorable. The application further provides a set of standardized and repeatable modeling, simulation, analysis and free energy calculation method. The application can clearly reveal the direct action mechanism of the evonik secondary alkaline and the AMPK, provides a reliable structural basis for target confirmation, structure optimization and drug research and development, and has important scientific value and application prospect.

Description

1. Technical Field

[0001] This invention belongs to the fields of computer-aided drug design, structural bioinformatics, and computational pharmacology. Specifically, it relates to a precise binding structure of a natural small molecule compound to a target protein, particularly a binding model of evodiamine and AMPK protein based on molecular docking and all-atom molecular dynamics simulations, as well as methods for constructing, optimizing, evaluating conformational stability, and calculating the binding free energy of this model. This invention can be used for target identification, molecular mechanism of action analysis, small molecule ligand structure optimization, and computer-aided drug screening and structure-activity relationship studies. 2. Background Technology

[0002] Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine / threonine protein kinase in eukaryotes. As a core regulatory molecule of cellular energy metabolism, AMPK senses changes in intracellular energy status and participates in regulating a series of key life processes, including glucose transport, glycolysis, fatty acid oxidation, lipid synthesis, mitochondrial biogenesis, and redox balance. The structural stability and functional activity of AMPK directly affect cellular energy homeostasis, and its functional abnormalities are closely related to the disruption of various metabolic-related physiological processes, thus making it an important research target in the fields of life sciences and drug development.

[0003] Natural small molecule compounds are an important source for discovering novel target regulators due to their diverse structures, wide availability, and good biocompatibility. Rutecarpine is an indolequinazoline alkaloid monomer isolated from traditional medicinal plants. Studies have shown that it has various biological activities, but whether it interacts directly with AMPK protein, its binding site, binding mode, binding affinity, and dynamic conformational stability remain unclear.

[0004] Current research has the following obvious shortcomings: (1) There is a lack of structural biological evidence for the interaction between Evodia rutaecarpa and AMPK, making it impossible to determine whether it is a direct binding; (2) The binding details such as key amino acids, hydrogen bonds, and hydrophobic interactions are not clearly defined, and the molecular mechanism is unclear; (3) There is a lack of stable binding models that have been verified by molecular dynamics over a long period of time, which cannot provide a reliable structural basis for mechanism research; (4) The binding mode has not been systematically evaluated by thermodynamics and kinetics, making it difficult to determine the binding strength, stability, and main driving force.

[0005] Therefore, constructing an accurate, reliable, repeatable, and long-term molecular dynamics-verified model of the binding of Evodia rutaecarpa alkaloid-AMPK, and clarifying its binding site, interaction type, conformational stability, and binding free energy, is of great scientific value and application prospect for revealing the interaction mechanism between natural small molecules and targets, and promoting target confirmation and lead compound structure optimization. 3. Summary of the Invention

[0006] 3.1 Purpose of the Invention

[0007] To address the gaps in existing technologies, this invention aims to provide a precise binding model of evodiamine and AMPK protein, along with a complete, standardized, and reproducible method for construction and multi-dimensional evaluation. This invention, for the first time, reveals the binding mode, key action sites, conformational stability, and binding thermodynamic characteristics of evodiamine and AMPK at the atomic level, filling a research gap in this field and providing a reliable structural basis for subsequent target mechanism research, small molecule structure optimization, and computer-aided drug design.

[0008] 3.2 Technical Solution

[0009] 3.2.1 Evodia rutaecarpa-AMPK binding model

[0010] This invention provides a stable binding model of evodiamine and AMPK protein, characterized in that:

[0011] 1. Evodia rutaecarpa binds to the active pocket region of the AMPK protein with high affinity, exhibiting a well-defined binding orientation, conformational convergence, and high spatial matching.

[0012] 2. Evodia rutaecarpa forms a stable intermolecular hydrogen bond with the Val98 residue of AMPK, with a bond length of 3.24 Å, which is the key force for the specific recognition of the ligand and the target.

[0013] 3. Evodia rutaecarpa is encapsulated by a hydrophobic pocket composed of residues such as Ala45, Lys47, Tyr97, and Leu148, forming a continuous, stable, and extensive network of hydrophobic interactions;

[0014] 4. Molecular docking results show that the binding free energy is -10.0 kcal / mol, indicating that the binding process can proceed spontaneously and has excellent affinity;

[0015] 5. Verified by 100 ns all-atom molecular dynamics simulation, the complex backbone RMSD is stably converged, the RMSF of key binding residues is significantly reduced, and the binding mode is highly conserved throughout the process.

[0016] 6. MM / PBSA binding free energy calculations show that the total binding free energy is -35.73 kcal / mol, with van der Waals interactions being the main driving force and hydrogen bonds providing specific recognition.

[0017] 3.2.2 Combined Model Construction Methods

[0018] A method for constructing an eugenol-AMPK binding model includes the following steps:

[0019] 1. Structural preparation: Obtain the three-dimensional structure of AMPK protein and the small molecular structure of Evodia rutaecarpa alkaloid, and perform hydrogenation, charge calculation, water molecule removal, and optimization of polar hydrogen and rotatable bonds;

[0020] 2. Molecular docking: AutoDock Vina was used to set up docking grid boxes in the AMPK active pocket region to complete global docking and local optimization, and the optimal conformation with the lowest binding free energy was selected;

[0021] 3. Simulation System Construction: The optimal docking complex was placed in a TIP3P water model dodecahedral box, and [the following was added]... Counteracting ions neutralize system charges; proteins are subjected to an Amber14SB force field, and small molecules are subjected to a GAFF force field.

[0022] 4. Energy Minimization: The conjugate gradient method is used to fully minimize energy, eliminate atomic spatial conflicts, and bring the system to the lowest energy state.

[0023] 5. System balancing: Perform NVT balancing and NPT balancing sequentially to stabilize temperature, pressure, density, and solvent distribution;

[0024] 6. All-atom molecular dynamics simulation: Perform a 100 ns production simulation and collect trajectory data;

[0025] 7. Conformational stability analysis: including RMSD, RMSF, radius of gyration Rg, solvent accessible surface area SASA, number of hydrogen bonds, and secondary structure stability analysis;

[0026] 8. Free Energy Landscape Analysis: Conformation distribution and thermodynamic stability are analyzed through principal component analysis and free energy landscape (FEL) analysis;

[0027] 9. Binding free energy and residue contribution calculation: The total binding free energy was calculated using the MM / PBSA method, and the amino acid residue contribution was decomposed to determine the key binding sites.

[0028] 3.2.3 Model Applications

[0029] The combination model of the present invention can be used for:

[0030] (1) To reveal the direct molecular mechanism of the interaction between evodiamine and AMPK;

[0031] (2) As a structural template for AMPK-targeted small molecule structure optimization;

[0032] (3) Used for computer-aided virtual screening, pharmacophore model construction and binding mode prediction;

[0033] (4) Used to elucidate the structure-activity relationship and target selectivity between natural small molecules and targets.

[0034] 3.3 Beneficial Effects

[0035] Compared with the prior art, the present invention has the following outstanding advantages and significant progress:

[0036] 1. First-ever establishment of a precise binding model: This invention is the first to construct a stable binding model between Evodia rutaecarpa and AMPK at the atomic level, clarifying the key binding sites and the types of interactions, filling a gap in the field;

[0037] 2. High affinity and specificity: The binding free energy is as low as -10.0 kcal / mol, the hydrogen bond and hydrophobic network are well defined, and it has excellent target recognition ability;

[0038] 3. Highly stable and reliable conformation: Validated by 100 ns molecular dynamics, the complex exhibits stable RMSD convergence, reduced flexibility of key residues, and conserved binding mode throughout the process, making the model realistic and reliable.

[0039] 4. Sufficient thermodynamic data and clear mechanism: The binding free energy of MM / PBSA is -35.73 kcal / mol, clearly indicating that van der Waals forces are the main driving force and hydrogen bonds are the specific recognition force;

[0040] 5. Standardized, reproducible, and scalable method: This invention provides a complete modeling process, applicable to the study of the binding mechanism between various natural small molecules and target proteins;

[0041] 6. Outstanding scientific value: It provides a key structural basis for AMPK target confirmation, the elucidation of the mechanism of action of natural small molecules, and the optimization of lead compounds, and has important scientific research value and application prospects. 4. Description of the attached drawings

[0042] Figure 1. Schematic diagram of the optimal binding mode of Evodia rutaecarpa alkaloids to AMPK protein molecules;

[0043] Figure 2. Comparison curve of RMSD between Evodia rutaecarpa alkaloid-AMPK complex and pure protein backbone;

[0044] Figure 3. Comparison of Evodia rutaecarpa alkaloid-AMPK complex and purified protein amino acid residue RMSF;

[0045] Figure 4. Comparison of radius of gyration (Rg) between the evodiamine-AMPK complex and pure protein;

[0046] Figure 5. Comparison of solvent-accessible surface area (SASA) of Evodia rutaecarpa-AMPK complex and pure protein;

[0047] Figure 6. Comparison of the number of hydrogen bonds inside the Evodia rutaecarpa-AMPK complex and the purified protein;

[0048] Figure 7. Changes in the number of protein-ligand hydrogen bonds during the simulation of the Evodia rutaecarpa-AMPK complex;

[0049] Figure 8. Free energy landscape (FEL) diagram of the pure AMPK protein system;

[0050] Figure 9. Free energy landscape (FEL) diagram of the Evodia rutaecarpa-AMPK complex system;

[0051] Figure 10. DSSP analysis diagram of the secondary structure of the Evodia rutaecarpa-AMPK complex;

[0052] Figure 11. Decomposition diagram of the binding free energy and amino acid residue contribution of the Evodia rutaecarpa-AMPK complex MM / PBSA.

[0053] Abbreviations: RMSD: Root Mean Square Deviation, RMSF: Root Mean Square Fluctuation, SASA: Solvent Accessible Surface Area, NVT: Canonical Ensemble, NPT: Isothermal and Isobaric Ensemble, PCA: Principal Component Analysis, FEL: Free Energy Landscape, MM: Molecular Mechanics, PBSA: Poisson-Boltzmann Surface Area Method, ATP: Adenosine Triphosphate, AMPK: Adenosine Oxide-Activated Protein Kinase, EC: Enzyme Committee Number, VDW: Van der Waals Force, HBA: Hydrogen Bond Acceptor, HBD: Hydrogen Bond Donor. 5. Detailed Implementation

[0054] 5.1 Experimental Materials and Tools

[0055] The AMPK protein structure was obtained from the RCSB PDB database; the Evodia rutaecarpa alkaloid structure was obtained from the PubChem database; simulation and analysis software included Gromacs 2024.1, AutoDock Vina, PyMOL, and LigPlot+; the Amber14SB force field was used for proteins, the GAFF force field was used for small molecules, and the TIP3P water model was used.

[0056] 5.2 Molecular docking

[0057] The protein and ligand were pretreated using AutoDockTools 1.5.6, which included adding polar hydrogen, calculating Gasteiger charge, and defining rotatable bonds. A docking box was set up in the AMPK active region, and AutoDock Vina was run to complete molecular docking. The conformation with the lowest binding free energy was selected as the optimal initial model. PyMOL and LigPlot+ were used to analyze hydrogen bonds, hydrophobic interactions, and binding modes.

[0058] The results showed that the binding free energy of evodiamine to AMPK was -10.0 kcal / mol, and it could spontaneously bind to the active pocket of the protein and form a 3.24 Å hydrogen bond with Val98. At the same time, it formed a stable hydrophobic network with Ala45, Lys47, Tyr97, Leu148, and Val98.

[0059] The experimental results are shown in Figure 1.

[0060] 5.3 Molecular Dynamics Simulation

[0061] The complex system was placed in a dodecahedral water box, with the box boundary 10 Å from the protein surface; [Addition] The system was made electrically neutral; energy minimization, NVT equilibration (200 ps), and NPT equilibration (200 ps) were performed sequentially; hydrogen bonds were constrained using the LINCS algorithm, and long-range electrostatic interactions were handled using the PME method, with a time step of 2 fs; a 100 ns production simulation was performed and the trajectory was saved.

[0062] The results show that the system temperature, pressure, and potential energy all tend to stabilize without abnormal fluctuations, indicating that the simulation process is reliable and effective.

[0063] The experimental results are shown in Figures 2 to 11.

[0064] 5.4 Conformation and Stability Analysis

[0065] (1) Root Mean Square Deviation (RMSD) Analysis

[0066] The RMSD of the protein backbone was calculated, and the results showed that the average RMSD of the complex system was 0.4417±0.0384 nm, which was lower than that of the pure protein system (0.4554±0.0595 nm), with less fluctuation and more stable conformation.

[0067] The experimental results are shown in Figure 2.

[0068] (2) Root Mean Square Fluctuation (RMSF) Analysis

[0069] Analysis of amino acid residue flexibility revealed that the RMSF values ​​of key residues Ala45, Lys47, Tyr97, Val98, and Leu148, which bind to Evodia rutaecarpa alkaloids, were significantly reduced, indicating more stable local conformations.

[0070] The experimental results are shown in Figure 3.

[0071] (3) Analysis of radius of gyration Rg

[0072] The overall compactness of the protein was evaluated by the radius of gyration. The results showed that the average Rg of the pure protein was 2.8993±0.0258 nm, and that of the complex was 2.9044±0.0217 nm, with no significant difference, and the structure remained compact and intact.

[0073] The experimental results are shown in Figure 4.

[0074] (4) Solvent-accessible surface area (SASA) analysis

[0075] SASA results showed that the average size of the pure protein was 310.87±4.44 nm², and that of the complex was 313.87±5.03 nm², with a difference of only 0.96% and no obvious structural swelling.

[0076] The experimental results are shown in Figure 5.

[0077] (5) Analysis of the number of hydrogen bonds inside the protein

[0078] The results showed that the pure protein had an average of 465.89±11.25 hydrogen bonds, while the complex had 459.68±10.02 hydrogen bonds. The protein remained stable overall, but local adaptive conformational adjustments occurred.

[0079] The experimental results are shown in Figure 6.

[0080] (6) Protein-ligand hydrogen bond analysis

[0081] Hydrogen bond statistics show that the complex retains an average of 0.9768 hydrogen bonds in a 100 ns simulation, mainly formed by Val98 stabilization, and the binding mode is highly conserved.

[0082] The experimental results are shown in Figure 7.

[0083] (7) Free Energy Landscape FEL Analysis

[0084] The results showed that the pure protein exhibited two dispersed low-energy conformations, while the complex exhibited a single, concentrated low-energy conformation, with significantly improved thermodynamic stability.

[0085] The experimental results are shown in Figures 8 and 9.

[0086] (8) Secondary structure DSSP analysis

[0087] DSSP analysis showed that the complex remained stable throughout the simulation. spiral, The secondary structures, such as folds, remain stable and continuous, without obvious unwinding or breakage.

[0088] The experimental results are shown in Figure 10.

[0089] 5.5 Calculations based on free energy

[0090] The mean binding free energy was calculated using the MM / PBSA method, and energy and residue decomposition were performed. The results showed that the total binding free energy was -35.73 kcal / mol, with van der Waals interactions being the main driving force; residue decomposition showed that Tyr97 and Val98 contributed the most significantly.

[0091] The experimental results are shown in Figure 11.

Claims

1. A model for the binding of evodiamine to AMPK protein, characterized in that, Evodiamine binds to the active pocket region of the AMPK protein, forming a stable hydrogen bond with the Val98 residue and a hydrophobic interaction network with the Ala45, Lys47, Tyr97, and Leu148 residues; the molecular docking binding free energy is -10.0 kcal / mol; the MM / PBSA binding free energy is -35.73 kcal / mol.

2. The combined model according to claim 1, characterized in that, Verified by 100 ns all-atom molecular dynamics simulations, the protein backbone of the binding model showed RMSD convergence and conformational stability, and the binding mode remained conserved throughout the simulation.

3. The combined model according to claim 1, characterized in that, Evodiamine forms a hydrogen bond with Val98 of AMPK with a bond length of 3.24 Å, which constitutes the key force for the specific recognition of the ligand and the target.

4. The combined model according to claim 1, characterized in that, Evodiamine significantly reduces the RMSF value of residues in the AMPK binding pocket region, constraining local conformational flexibility and enhancing protein structural stability.

5. The combined model according to claim 1, characterized in that, The radius of gyration Rg and solvent-accessible surface area SASA of the complex remained stable during the simulation, and the overall protein structure was compact without significant swelling or unfolding.

6. The combined model according to claim 1, characterized in that, During the simulation, approximately one hydrogen bond was stably maintained between Evodia rutaecarpa and AMPK, indicating a high hydrogen bond occupancy rate and a strong bond.

7. The combined model according to claim 1, characterized in that, Free energy landscape (FEL) analysis revealed that the complex exhibited a single, concentrated, low-energy conformation well, with significantly better thermodynamic stability than the pure protein.

8. A method for constructing an eugenol-AMPK protein binding model, characterized in that, Includes the following steps: (1) Preparation and pretreatment of protein and small molecule structures; (2) Molecular docking to obtain the optimal binding conformation; (3) Construct a solvation molecular dynamics system; (4) Energy minimization and system equilibrium; (5) 100 ns all-atom molecular dynamics simulation; (6) Conformational stability and hydrogen bond analysis; (7) Free energy landscape analysis; (8) MM / PBSA binding free energy calculation and residue contribution decomposition.

9. The method according to claim 8, characterized in that, Stability analysis included RMSD, RMSF, Rg, SASA, number of hydrogen bonds, secondary structure DSSP, and free energy landscape (FEL) analysis.

10. The use of the combined model as described in claim 1, characterized in that, Used for AMPK target mechanism research, optimization of natural small molecule ligand structures, computer-aided drug screening, or pharmacophore model construction.

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