Molecular simulation method for designing and screening oligopeptide drug carrier with efficient transmembrane transport capability
Cell membrane models with different structures were constructed through molecular dynamics simulation methods, and the interaction between short peptides and cell membranes was studied, which solved the problem of difficult to efficiently screen short peptides with efficient transcellular membrane transport capabilities in the prior art, achieving simple and efficient screening and design of drug carriers.
Patent Information
- Application Number
- CN202510092247.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-16
AI Technical Summary
It is difficult to efficiently screen short peptides with efficient transcellular membrane transport capability in the prior art, and the experimental methods have problems such as cumbersome operation, long cycle and high cost.
Molecular dynamics simulation method was used to construct a cell membrane model composed of phospholipids of different structures based on GROMACS, and the mechanism of its transcellular membrane transport ability was studied by simulating the interaction between short peptides and cell membranes.
In-depth exploration and screening of the transcellular membrane transport capacity of short peptides has been achieved, the operation process has been simplified, the cost and time has been reduced, and a convenient and effective way to design and screen drug carriers.
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Figure CN120015170A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular simulation, and in particular to a molecular simulation method for designing and screening a short peptide with high-efficiency transcellular membrane transport capability as a drug carrier. Background Art
[0002] Short peptides are a class of compounds formed by amino acids connected by peptide bonds. They have the characteristics of small molecular weight, simple structure, easy synthesis and modification, etc. Therefore, they have great advantages in the design and screening of drug carriers. In addition, short peptides have good biocompatibility and degradability, which can effectively reduce side effects and improve drug delivery efficiency. Currently, two natural short peptides such as cell penetrating peptides and antimicrobial peptides have also achieved a series of important application results in clinical treatment. Short peptides as drug carriers are being valued by more and more researchers in delivering drugs within cells. Therefore, screening short peptides with efficient transcellular membrane transport capabilities has important biological significance.
[0003] Phospholipids are the main components of cell membranes, and their general form is a hydrophilic head connected to two hydrophobic hydrocarbon tails. Different cell membrane models can be constructed according to the different phospholipid components that make up the cell membrane. The physical properties of the membrane and the interaction between phospholipids will cause different phenomena in the interaction process with short peptides. Although experimental techniques have been continuously developed and improved in recent years, providing an unprecedented perspective for the microscopic structure observation of the interaction process between cell membranes and other substances and the extraction of phospholipid arrangement characteristics, the screening of short peptides with efficient transcellular membrane transport capabilities using conventional experimental methods is difficult, costly, and time-consuming, and a new technical method is urgently needed. Summary of the invention
[0004] In view of this, the purpose of the present invention is to propose a molecular simulation method for the design and screening of short peptides with efficient transcellular membrane transport ability as drug carriers. The method is simple and easy to operate. The computational technology of studying the time evolution of microscopic systems under classical mechanics by numerical simulation methods is used. The present invention adopts molecular dynamics simulation methods, based on GROMACS, to construct a cell membrane model composed of phospholipids with different structures and a simulated environment inside and outside the cell, and systematically explores the mechanism of the transcellular membrane transport ability of short peptides.
[0005] To achieve the above object, the technical solution of the present invention is achieved as follows: A molecular simulation method for screening short peptides with efficient transcellular membrane transport ability, the method comprising the following steps: (1) Construction of cell membrane models with different phospholipid components: Use the CHARMM-GUI online website to build: select phospholipids with different structures according to the head group, tail chain length, and hydrocarbon chain unsaturation, use the CHARMM-GUI website to build a small module membrane system with different phospholipid combinations, and then use GROMACS to expand the small module membrane system to the required size, or use Material Studio and other software to build: model in Material Studio according to the selected phospholipid structure, and use Automated Topology Builder to generate itp files, and then use Genmixmem to combine phospholipids with different structures to achieve a cell membrane model of the required size; (2) Construction of short peptide models with different structures: Determine the amino acid sequence of the short peptide and use AlphaFold or Pymol to model the short peptide to obtain the pdb file. Then use GROMACS to process the short peptide structure file using pdb2gmx and select the appropriate force field to obtain the itp file. (3) Simulating the kinetics of short peptide transport across cell membranes: Using GROMACS, the short peptide and the cell membrane are placed in a box of appropriate size, solvent and ions are added, and the simulation is performed for a sufficient time to fully simulate the system until the short peptide is adsorbed on the cell membrane or embedded in the cell membrane; (4) Mechanism analysis: VMD visualization software is used to capture the dynamic process and details of short peptides crossing the membrane, and VMD's built-in script is used to analyze the overall evolution rate and change behavior of short peptides during the membrane crossing process.
[0006] Furthermore, the phospholipid components can be freely combined to form different cell membrane models as needed. At the same time, the force field and temperature settings can be selected as needed to simulate the temperature conditions in different environments. The initial cell membrane model is subjected to energy minimization, pre-equilibrium simulation and equilibrium simulation to obtain a stable cell membrane model. After obtaining a stable cell membrane model,
[0007] Furthermore, a short peptide was placed in the simulation box and a 5 nm water layer and ions were added to both leaves of the cell membrane. Then, energy minimization, pre-equilibrium simulation, and equilibrium simulation were performed to obtain a stable model.
[0008] Furthermore, energy minimization was performed using two 5000-step steepest descent methods; the pre-equilibrium simulation was performed five times in an isothermal and isobaric ensemble, with each time being a 1 ns pre-equilibrium, with the time step gradually increasing and the position constraints on the head group reduced in each pre-equilibrium; the equilibrium simulation before adding the short peptide was performed for 200 ns in the NPT ensemble, using the Parrinello-rahman pressure control method and V-rescale thermal bath; the equilibrium simulation after adding the short peptide was performed for 1 ns in the NPT ensemble, using the Parrinello-rahman pressure control method and V-rescale thermal bath.
[0009] Compared with the prior art, the molecular simulation method for studying the design and screening of short peptides with efficient transcellular membrane transport ability as drug carriers described in the present invention has the following advantages: (1) The molecular simulation method for studying the structure of short peptides and cell membrane structure for the design and screening of short peptides as drug carriers described in the present invention can realize the use of different phospholipid structures for cell membrane modeling and transmembrane process simulation, overcome the difficulty of analyzing the molecular dynamic details, and realize the correspondence between structural differences and free energy barrier differences, meeting the quantitative investigation needs of phospholipid structure action mechanism analysis. The method is simple to operate and has strong applicability. The phospholipid composition and membrane component composition are adjustable. The short peptide structure can be designed and modeled as needed, which is suitable for the reproduction of various short peptides and biological cell membrane systems, making up for the shortcomings of cumbersome operation, long cycle and high cost in experimental research. In general, the present invention can deeply explore and screen short peptides with efficient transcellular membrane transport ability and potential as drug carriers, providing a convenient and effective way for the design and exploration of short peptide drug carriers. (2) The present invention analyzes the cell transport ability of short peptides from the overall cell membrane structure. Since the transmembrane of short peptides is highly correlated with the physical properties of the membrane, the thickness of the inner and outer membranes of the cell membrane, the rigidity and flexibility of the membrane, etc. need to be quantitatively measured. Through the script analysis tools such as FATSLiM, membrane fluctuation calculation and GROMACS built-in analysis modules (gmx density, etc.), the rigidity and flexibility of the membrane and a series of physical properties were obtained. At the same time, in the transmembrane stage, short peptides will frequently contact the head and tail structures of phospholipids, so it is also necessary to quantitatively analyze the individual area (APL) of phospholipids, thickness changes, tail chain insertion performance, head group contact times, and phospholipid arrangement order. Through the above analysis and combined with free energy barrier analysis, the transport capacity of short peptides with different structures to cell membranes with different structures is summarized. This analysis deepens the research on the cellular transport process of short peptides and analyzes the mechanism of the influence of different structures of short peptides and phospholipids on cellular transport from a rare perspective. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 A flow chart of the rights involved in this patent; Figure 2 The schematic diagram of the initial configuration of the short peptide transcellular membrane transport simulation system shows the components of the short peptide and cell membrane system, including ions for balancing charges, short peptides, and plasma membranes composed of different phospholipids, in which water molecules have been hidden and the box size is marked by a blue solid line. In particular, different ion components and phospholipid components can be combined and constructed as needed; Figure 3 Schematic diagrams of simulations under different sodium ion concentrations, (a) is a schematic diagram of the simulation system at 0 ns and 10 ns under the condition of no sodium ions, (b) is a schematic diagram of the simulation system at 0 ns and 10 ns under the condition of 5 sodium ions; Figure 4 Schematic diagrams of simulations under different numbers of short peptides, (a) is a schematic diagram of a simulation system with one short peptide at 0 ns and 10 ns, (b) is a schematic diagram of a simulation system with four short peptides at 0 ns and 10 ns; Figure 5 Schematic diagrams of simulations under different chloride ion concentration differences, (a) is a schematic diagram of the simulated final configuration when there are 3 chloride ions in the intracellular environment, (b) is a schematic diagram of the simulated final configuration when there are 5 chloride ions in the intracellular environment; Figure 6 The density distribution diagrams of the simulated final configurations under different chloride ion concentration differences, (a) is the density distribution diagram of the simulated final configuration when there are 3 chloride ions in the intracellular environment, (b) is the density distribution diagram of the simulated final configuration when there are 5 chloride ions in the intracellular environment; Figure 7 Schematic diagram of the simulated hydrogen bond numbers under different chloride ion concentration differences, (a) is a schematic diagram of the simulated hydrogen bond numbers when there are 3 chloride ions in the intracellular environment, and (b) is a schematic diagram of the simulated hydrogen bond numbers when there are 5 chloride ions in the intracellular environment. DETAILED DESCRIPTION
[0011] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.
[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, and the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.
[0013] All molecular dynamics simulations involved in the present invention were completed using the open source simulation software GROMACS2020.6, modeling was completed using CHARMM-GUI or using software such as Material Studio, visualization analysis was performed using VMD 1.9.3 software, and post-data processing and analysis was performed using GROMACS software.
[0014] Example 1 Construction of cell membrane models with different structural phospholipid components In order to better understand the mechanism of the influence of phospholipid structure on transmembrane process, cell membranes with different structural phospholipid components were constructed to reflect the compositional diversity of biological membranes.
[0015] The membrane layer of the cell membrane includes dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), etc. The selection of specific phospholipid types and proportions can be determined according to the needs of the user. This example only takes the simplest 100% DPPC phospholipid membrane as an example. The workflow is as follows Figure 1 shown.
[0016] CHARMM-GUI method: First, use the membrane layer construction module on the CHARMM-GUI website to select the appropriate system ratio, fix the number of phospholipids in the upper and lower layers, set the appropriate system temperature to 310K, and construct a small module membrane system. Then, use the GROMACS built-in command to expand the small module to the required size of the system. In this example, the small membrane block is expanded by 2 times in the X and Y directions.
[0017] Material Studio method: First, use Material Studio to build a DPPC molecular model, use the ATB website to generate an itp file, and then use Genmixmem software to select an appropriate system ratio, fix the number of phospholipids in the upper and lower layers, and then build a small module membrane system. After that, use the GROMACS built-in command to expand the small module to the required size of the system. In this example, the small membrane block is expanded by 2 times in the X and Y directions.
[0018] When balancing the cell membrane, no water layer is added above and below the cell membrane, which is periodic. The initial cell membrane model is constructed and a 20ns equilibrium simulation is performed under the NPT ensemble.
[0019] After obtaining a stable cell membrane model, determine the amino acid sequence of the short peptide and use AlphaFold or Pymol to model the short peptide, obtain the pdb file, and then use GROMACS to process the short peptide structure file with pdb2gmx and select the appropriate force field to obtain the itp file. Then, put the short peptide into the already stable cell membrane model, add a 5nm water layer and ions above and below the cell membrane, and then perform the same energy minimization and pre-equilibrium simulation, complete a 1ns equilibrium simulation, and obtain the short peptide-cell membrane model, such as Figure 2 shown.
[0020] Example 2: Using GROMACS to simulate and analyze short peptide transcellular membrane behavior
[0021] 1. Simulate short peptide transmembrane behavior When simulating the short peptide-cell membrane system, the simulation time step is set to 2fs. The number of simulation steps depends on the needs. In this example, it is set to 100,000,000 steps. Use GROMACS to perform dynamic simulations and record data every 500,000 steps to generate xtc, trr and other data files for subsequent analysis. The above parameters can be modified as needed. For example, by modifying parameters such as ion concentration and the number of short peptides, different systems can be obtained, such as Figure 3 , Figure 4 and Figure 5 shown.
[0022] 2. Data Analysis The VMD visualization software can be used to capture the dynamic process and details of membrane splitting. The Tcl script provided by VMD can be used to analyze the overall evolution rate and change behavior during the membrane splitting process. In order to compare the effects of various structures on the transmembrane energy barrier, the gmx hbond and density modules in GROMACS were used to analyze the dynamic simulation results. The hydrogen bond number and density distribution at different stages were compared, and the key factors were extracted in combination with the dynamic details to provide analysis direction for subsequent mechanism interpretation.
[0023] Figure 6 It is the density distribution diagram of the simulated final configuration under different chloride ion concentration differences, which refers to the density distribution of different components in the Z-axis direction. Through the density distribution diagram, it can be observed that when there are 3 chloride ions in the intracellular environment, the water molecules are discontinuously distributed in the Z-axis direction of 0-16nm, among which they are isolated by phospholipid components at 4.5nm and 12nm. At the same time, the short peptides are concentrated at the head of the phospholipid (2nm) and fail to pass through the phospholipid head during the simulation time. When there are 5 chloride ions in the intracellular environment, the water molecules are continuously distributed from 0nm to 10nm in the Z-axis direction, breaking through the limitation of phospholipids. This is because the short peptides pass through the phospholipid head and enter the hydrophobic area of the plasma membrane, that is, 2.5nm to 5nm.
[0024] Figure 7 The figure is a schematic diagram of the number of hydrogen bonds simulated under different chloride ion concentration differences. When there are 3 chloride ions in the intracellular environment, the number of hydrogen bonds bound within 0.35nm is large. When there are 5 chloride ions in the intracellular environment, the number of hydrogen bonds bound within 0.35nm decreases. At the same time, the total amount of hydrogen bonds does not change significantly. To summarize the above rules, positively charged short peptide structures are more likely to achieve transmembrane and cell transport. The characteristics of transmembrane and cell transport are as follows: First, the short peptide gradually moves closer to the cell membrane in the extracellular environment, and finally adsorbs on the cell membrane and fits the cell membrane surface. Secondly, the hydrophobic domain of the short peptide breaks through the head of the cell membrane and penetrates into the cell membrane. The short peptide gradually moves into the cell membrane and crosses the membrane. On this basis, the greater the membrane potential, that is, the greater the ion concentration difference between the intracellular and extracellular environments, the easier it is to promote the transmembrane and cell transport of short peptides. At the same time, the more short peptides there are, the easier it is to observe the transmembrane phenomenon.
[0025] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A molecular simulation method for designing and screening polypeptide small molecule drug carriers with efficient transcellular membrane transport capability, the method comprising the following steps: (1) Construction of cell membrane models with different phospholipid components: Use the CHARMM-GUI online website to build: select phospholipids with different structures according to the head group, tail chain length, and hydrocarbon chain unsaturation, use the CHARMM-GUI website to build a small module membrane system with different phospholipid combinations, and then use GROMACS to expand the small module membrane system to the required size, or use Material Studio and other software to build: model in Material Studio according to the selected phospholipid structure, and use Automated Topology Builder to generate itp files, and then use Genmixmem to combine phospholipids with different structures to achieve a cell membrane model of the required size; (2) Construction of short peptide models with different structures: Determine the amino acid sequence of the short peptide and use AlphaFold or Pymol to model the short peptide to obtain the pdb file. Then use GROMACS to process the short peptide structure file using pdb2gmx and select the appropriate force field to obtain the itp file. (3) Simulating the kinetics of short peptide transport across cell membranes: Use GROMACS to place the short peptide and the cell membrane in a box of appropriate size, add solvent and ions, and simulate for a sufficient time to fully simulate the system until the short peptide is adsorbed on the cell membrane or embedded in the cell membrane; (4) Mechanism and visualization analysis: VMD visualization software is used to capture the dynamic process and details of short peptides crossing the membrane, and VMD's own script is used to analyze the visualization of the overall dynamic process of short peptides crossing the membrane. In addition, GROMACS software can be used to analyze the dynamic curves related to short peptides and cell membranes.
2. The molecular simulation method for studying the effect of phospholipid structure on biomembrane fission according to claim 1, characterized in that: The phospholipid components can be freely combined as needed to form different cell membrane models. At the same time, the force field and temperature settings can be selected as needed to simulate the temperature conditions in different environments. The initial cell membrane model is subjected to energy minimization, pre-equilibrium simulation and equilibrium simulation to obtain a stable cell membrane model. After obtaining the stable cell membrane model, a short peptide is added and a 5nm water layer and ions are added to the two leaves of the cell membrane. Energy minimization, pre-equilibrium simulation and equilibrium simulation are then performed to obtain a stable model.
3. The molecular simulation method for studying the effect of phospholipid structure on biomembrane fission according to claim 2, characterized in that: Energy minimization was performed using two 5000-step steepest descent methods; the pre-equilibrium simulation was performed five times in an isothermal and isobaric ensemble, with each time being 1 ns, with the time step gradually increasing and the position constraints on the head group reduced in each pre-equilibrium; the equilibrium simulation before adding the short peptide was performed for 200 ns in the NPT ensemble, using the Parrinello-rahman pressure control method and V-rescale thermal bath; the equilibrium simulation after adding the short peptide was performed for 1 ns in the NPT ensemble, using the Parrinello-rahman pressure control method and V-rescale thermal bath.
4. An application of a molecular simulation method for studying the transcellular membrane transport ability of short peptides as described in any one of claims 1 to 3 in studying the movement laws and microscopic mechanisms of short peptides across cell membranes from a microscopic perspective.