High-throughput screening method for enzyme-responsive antitumor drug delivery polymeric carriers

Abstract

The application discloses a high-throughput screening method of an enzyme-responsive anti-tumor drug delivery polymer carrier. The method comprises the following steps: performing geometry optimization and calculating atomic charges on each candidate enzyme-responsive polymer carrier monomer and a substituted structure thereof based on a density functional theory (DFT) first principle to develop a corresponding all-atom molecular force field; constructing a force field of the enzyme-responsive polymer in batches based on the atomic molecular force field; researching a binding mode and a stable conformation of the enzyme-responsive polymer carrier and a protease, a thermodynamic behavior and a stable conformation of the enzyme-responsive polymer carrier folding, a binding strength of the enzyme-responsive polymer carrier and the protease and a catalytic efficiency of the enzyme-responsive polymer carrier and the protease; and screening the enzyme-responsive polymer carrier suitable for anti-tumor drug delivery based on the research results. The method can be used as a new way for efficiently obtaining the enzyme-responsive anti-tumor drug delivery polymer carrier.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers. It comprehensively utilizes various computational methods and techniques to screen for enzyme-responsive antitumor drug delivery polymer carriers that respond to gamma-glutamyl transferase (GGT), aminopeptidase APN (a protein overexpressed in various tumors), activated protease FAP (an enzyme overexpressed in tumor fibroblasts), and matrix metalloproteinase MMP (a protein overexpressed in invasive tumors). Background Technology

[0002] Highly effective treatment of malignant tumors has always been a major need in my country. Despite the emergence of new therapies such as tumor immunotherapy, chemotherapy remains one of the main methods of clinical cancer treatment and plays an important adjunctive role among other treatments. However, its application is hampered by significant side effects and limited efficacy. Nanomedicine technology is an important means of reducing the toxic side effects of chemotherapy.

[0003] Domestic and international scholars have conducted extensive research on improving the clinical efficacy of nanomedicines. Analysis of the entire in vivo delivery process of nanomedicines—from blood to tumor accumulation, penetration, endocytosis, and intracellular drug release—has revealed that extravasation from tumor blood vessels and diffusion within tumor tissue are the two major bottlenecks hindering their clinical efficacy. Currently, the theoretical basis for nanomedicine accumulation within tumors remains the EPR effect, whereby nanomedicines passively diffuse from the blood into tumor tissue through the pores of the tumor capillary walls. However, recent studies have shown that the permeability of tumor capillaries in clinical patients is much weaker and exhibits strong heterogeneity (varying depending on tumor stage, location, cancer type, and patient), making it difficult for nanomedicines to extravasate from blood vessels into tumor tissue. Furthermore, tumor tissue possesses pathological characteristics such as high cell density, dense matrix, and high intratumoral pressure. The large size and low diffusion coefficient of nanomedicines make diffusion within the tumor extremely difficult, preventing drug delivery to cells far from blood vessels. These two bottlenecks result in the low clinical efficacy of nanomedicines and have led to the failure of many newly developed nanomedicines in clinical application.

[0004] The adsorption of carrier materials or nanomedicines onto the cell membrane is crucial for triggering endocytosis and subsequent transcytosis. In previous work, a collaborative team (Professor Shen Youqing of Zhejiang University) discovered that polymer-camptothecin conjugates, after being hydrolyzed by gamma-glutamyl transferase (GGT) on the cell membrane to produce amino groups and acquire a positive charge, can adsorb onto the cell membrane. The polymer-camptothecin conjugates then undergo rapid transcytosis-mediated endocytosis-exocytosis, facilitating transendothelial transport. Furthermore, they found that the polymer-camptothecin conjugates can be uniformly distributed in tumor tissues and exhibit highly efficient tumor-suppressive activity (Nature Nanotechnology (2019) 799–809). However, previous experiments characterizing the interaction between enzyme-responsive antitumor drug delivery polymer carriers and proteases were time-consuming and laborious, making it difficult to optimize and screen enzyme-responsive antitumor drug delivery polymers over a large spatial scale. Summary of the Invention

[0005] To address the shortcomings of the aforementioned background technologies, this invention provides a high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers. This method can accurately assess the binding strength between enzyme-responsive polymers and proteases during the rational optimization and design of enzyme-responsive antitumor drug delivery polymer carriers. It provides a computer-simulated method for calculating the affinity between enzyme-responsive polymers and proteases, which is more time- and labor-saving than experimental methods, and represents a new approach to obtaining highly efficient enzyme-responsive antitumor drug delivery polymer carriers.

[0006] The present invention adopts the following technical solution:

[0007] A high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers includes the following steps:

[0008] (i) Based on the first principles of density functional theory, the B3LYP functional +6‒31G basis set was selected to perform geometric optimization on the monomers and their substitution structures of each candidate enzyme response polymer carrier and to calculate their atomic charges, thereby developing the corresponding all-atom molecular force field.

[0009] (ii) Based on the all-atomic molecular force field of step (i), the force field of enzyme-responsive polymers is constructed in batches;

[0010] (iii) Molecular docking and molecular dynamics were used to study the binding mode of enzyme-responsive polymers and proteases; molecular dynamics simulations were performed based on the force field of the protease to study the stable conformation of enzyme-responsive polymers and proteases;

[0011] (iv) The thermodynamic behavior and stable conformation of enzyme-responsive polymer carrier folding were studied using enhanced sampling copy exchange molecular dynamics simulations;

[0012] (v) The binding strength between enzyme-responsive polymer carriers and proteases was compared and studied using a metakinetic method;

[0013] (vi) The catalytic efficiency of enzyme-responsive polymer carriers and proteases was studied by using a coupled quantum mechanics / molecular force field comparison.

[0014] (vii) Based on the calculation results of steps (iii)-(vi), efficient enzyme-responsive antitumor drug delivery polymer carriers are screened.

[0015] In the above technical solution, further, in step (i), the first-principles calculation of density functional theory is performed using GAUSSIAN software, the all-atomic molecular force field is the universal AMBER force field, and the software specifically used for constructing the all-atomic molecular force field is Ambertools.

[0016] Furthermore, the proteases in steps (iii), (v) and (vi) include gamma-glutamyl transferase (GGT), aminopeptidase APN which is highly expressed in various tumors, activated protease FAP which is overexpressed in tumor fibroblasts, and matrix metalloproteinase MMP which is highly expressed in invasive tumors. The force field of these enzymes adopts the AMBER14 force field.

[0017] Furthermore, in step (iv), the enhanced sampling copy exchange molecular dynamics simulation employs solute annealing copy exchange (REST).

[0018] Furthermore, in step (v), the meta-dynamics method employs well-tempered meta-dynamics simulation, which guarantees smooth convergence of the calculation results. Specifically, it is implemented using the GROMACS+Plumed software package.

[0019] This invention innovatively integrates multiple molecular computational simulation techniques to systematically study the multifaceted properties of polymer carriers and proteases, including binding strength and catalytic efficiency—key properties for enzyme-responsive antitumor drug delivery polymer carriers—by combining the advantages of different computational methods. This invention provides a computer-simulated computational method for predicting the affinity and catalytic efficiency of enzyme-responsive polymers and proteases, which is more time- and labor-saving than experimental methods. It can serve as a new and efficient approach to obtaining enzyme-responsive antitumor drug delivery polymer carriers.

[0020] This invention utilizes computer modeling of enzyme-responsive polymer carriers, employs enhanced sampling copy exchange molecular dynamics simulation to study the thermodynamic behavior and stable conformation of enzyme-responsive carrier folding, and uses a meta-dynamics algorithm to calculate the binding strength between enzyme-responsive polymer carriers and proteases. Furthermore, it employs coupled quantum mechanics / molecular force fields to achieve precise and general-purpose binding affinity screening of the enzyme-responsive polymer library, thereby designing and screening highly efficient enzyme-responsive antitumor drug delivery polymer carriers. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings;

[0022] Figure 1 It is a monomer for polymer carriers that deliver enzyme-responsive antitumor drugs;

[0023] Figure 2 This is a flowchart illustrating the construction process of the enzyme-responsive antitumor drug delivery polymer carrier monomer of the present invention;

[0024] Figure 3 This is a flowchart of the high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers of the present invention. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments.

[0026] Figure 1 The structure of R2 determines the enzyme's responsiveness and specificity, while the substituent R1 at the ortho position of the amide group strongly influences the enzyme's activity in catalyzing the hydrolysis of amides. Optional groups for R1 include methyl, ethyl, isopropyl, phenyl, benzyl, phenethyl, ethoxyphenyl, vinyl, ethynyl, acetyl, amino, carboxyl, hydroxyl, and nitro. Optional groups for R2 include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0027] Figure 2In this process, SMILES (a specification of molecular structure explicitly described using ASCII strings) strings were artificially constructed for enzyme-responsive polymer monomers. Then, the open-source software RDkit was used to convert the SMILES format polymer monomers into molecular files (SDF format) with three-dimensional structures. Pymol was then used to open and inspect the three-dimensional monomer files, removing hydrogen atoms in inappropriate positions according to the actual protonation state of the molecules under physiological pH conditions. First-principles calculations based on density functional theory (DFT) were performed on the polymer carrier monomers using GAUSSIAN 16, selecting the B3LYP functional + 6‒31G basis set. Ambertools was used to develop the corresponding all-atom molecular force field; specifically, the force field parameters used in the calculations were the universal AMBER force field (GAFF).

[0028] like Figure 3 This is a flowchart of the high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers of the present invention. First, the force field of the enzyme-responsive polymer is constructed. Second, for proteases, gamma-glutamyl transferase (GGT), aminopeptidases highly expressed in various tumors (APN), activated protease overexpressed in tumor fibroblasts (FAP), and matrix metalloproteinases highly expressed in invasive tumors (MMP) are downloaded from the PDB database and modeled. The binding strength between different polymers and proteases is calculated using the structure of the enzyme catalytic site combined with semi-flexible molecular docking technology. Polymers are ranked from strongest to weakest binding strength, and the predicted polymer-enzyme catalytic hydrolysis rate is calculated using QM / MM and metakinetic methods. Based on this, responsive polymers with corresponding enzyme specificity but different catalytic hydrolysis rates are synthesized, and their enzyme-catalyzed hydrolysis rates are determined by HPLC. The semi-flexible molecular docking technology is implemented using Autodock Vina, the metakinetic method is implemented using GROMACS and Plumed software, and the QM / MM method is implemented using GROMACS and CP2K software.

Claims

1. A high-throughput screening method for enzyme-responsive polymer carriers for antitumor drug delivery, characterized in that, Includes the following steps: (i) Based on the first principles of density functional theory, the B3LYP functional and 6‒31G basis set were selected to perform geometric optimization on the monomers and their substitution structures of each candidate enzyme response polymer carrier and to calculate their atomic charges, thereby developing the corresponding all-atom molecular force field. (ii) Based on the all-atom molecular force field constructed in step (i), the force field of enzyme-responsive polymers is constructed in batches; (iii) Molecular docking and molecular dynamics were used to study the binding mode of enzyme-responsive polymers and proteases; molecular dynamics simulations were performed based on the force field of the protease to study the stable conformation of enzyme-responsive polymers and proteases; (iv) The thermodynamic behavior and stable conformation of enzyme-responsive polymer carrier folding were studied using enhanced sampling copy exchange molecular dynamics simulations; (v) The binding strength between enzyme-responsive polymer carriers and proteases was compared and studied using a metakinetic method; (vi) The catalytic efficiency of enzyme-responsive polymer carriers and proteases was studied by using a coupled quantum mechanics / molecular force field comparison. (vii) Based on the calculation results of steps (iii)-(vi), highly efficient enzyme-responsive antitumor drug delivery polymer carriers were screened. In step (iv), the enhanced sampling copy exchange molecular dynamics simulation employs solute annealing copy exchange; In step (v), the meta-dynamics method employs well-tempered meta-dynamics simulation.

2. The high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers according to claim 1, characterized in that, In step (i), the all-atomic molecular force field is the universal AMBER force field.

3. The high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers according to claim 1, characterized in that, In step (iii), the force field of the protease is an AMBER14 force field.

4. The high-throughput screening method for enzyme-responsive antitumor drug delivery polymer carriers according to claim 1, characterized in that, The proteases in steps (iii), (v) and (vi) include gamma-glutamyl transferase (GGT), aminopeptidase (APN), activated protease (FAP), and matrix metalloproteinase (MMP).

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