A protein-based drug repositioning method and application

Abstract

The present application relates to a kind of protein-based drug relocation method and application, comprising the following steps: ① preparation of receptor protein beta-catenin: obtain beta-catenin protein three-dimensional structure from AlphaFold database, and carry out structure optimization processing;② preparation of ligand molecular library: from Pubchem database download FDA approved drug small molecule compound as ligand small molecule library, beta-catenin co-crystallization ligand inhibitor RS6452 small molecule is judged as reference standard screening ligand and incorporated into ligand library;③ setting docking active pocket: with the position of ligand in beta-catenin co-crystallization complex determine docking center coordinates, set docking radius;④ compound virtual screening: in virtual screening software setting docking times, the compound to be screened is docked into receptor protein active pocket, and the docking binding energy score is sorted, obtains the candidate compound with potential inhibition beta-catenin protein activity;⑤ molecular docking: using molecular docking software, the ligand small molecule with high docking binding energy score screened is docked with beta-catenin protein, and the best conformation of docking effect is obtained.The drug relocation method provided by the present application has the advantages of rapid, efficient, low cost, etc., the selected ligand library is FDA-approved drug, potential anti-hepatoblastoma inhibitors are found by virtual screening method, drug relocation is realized, drug development cycle can be shortened, economic cost can be reduced, and social benefits can be effectively improved.

Description

Technical Field

[0001] This invention relates to a drug repositioning method, and more specifically, to a virtual screening method for beta-catenin protein inhibitors, wherein the repositioned drug can prevent and / or treat beta-catenin protein-mediated hepatoblastoma disease. Background Technology

[0002] Hepatoblastoma is a malignant tumor of the liver that occurs in children, typically between 2 and 3 years of age. Extensive preclinical and clinical research has provided evidence that increased translocation of beta-catenin from the cell surface to the cytoplasm and nucleus, and its intracellular accumulation, are directly correlated with cancer severity. Therefore, alterations in beta-catenin and its target genes may serve as biomarkers for the diagnosis and prognosis of active tumors in children (Bell, D., Ranganathan, S., Tao, J. and Monga, SP(2017) Novel advances in understanding of molecular pathogenesis of hepatoblastoma: a Wnt / β-Catenin perspective. Gene Expr. 17, 141–154).

[0003] The Wnt / β-catenin signaling pathway plays a crucial role in controlling liver homeostasis, maintaining adhesion junctions, metabolic partitioning, and regeneration, suggesting its involvement in almost all relevant aspects of liver function (Russell, JO and Monga, SP (2018) Wnt / β-catenin signaling in liver development, homeostasis, and pathobiology. Annu. Rev. Pathol. 13, 351–378). However, its aberrant activation is associated with the development of various liver diseases, including hepatoblastoma (Perugorria, MJ, Olaizola, P., Labiano, I., Esparza-Baquer, A., Marzioni, M., Marin, JJG et al. (2019) Wnt-β-catenin signalling in liver development, health and disease. Nat. Rev. Gastroenterol. Hepatol. 16, 121–136). In fact, full-length beta-catenin has been reported to be a major factor influencing early liver development (Ranganathan, S., Tan, X. and Monga, SP (2005) β-Catenin and met deregulation in childhood hepatoblastomas. Pediatr. Dev. Pathol. 8, 435–447) (Salvatore, M., Lorenzetti, S., Maranghi, F., Mantovani, A. and Taruscio, D. (2008) Molecular link(s) between hepatoblastoma pathogenesis and exposure to di-(2-ethylhexyl)phthalate: a hypothesis. Folia Med. (Plovdiv) 50, 17–23).However, calpain-mediated cleavage of beta-catenin produces a 75-kDa truncated beta-catenin (lacking 95 N-terminal amino acids), which is transported to the cytoplasm and nucleus, inducing the development of hepatoblastoma (Lade, A., Ranganathan, S., Luo, J. and Monga, SP (2012) Calpain induces N-terminal truncation of β-catenin in normalmurine liver development: diagnostic implications in hepatoblastomas. J.Biol. Chem. 287, 22789–22798). Studies have shown that nuclear beta-catenin localization is directly related to poor differentiation, and its localization in embryonal and undifferentiated hepatoblastoma is significantly higher than that in fetal hepatoblastoma (Park, WS, Oh, RR, Park, JY, Kim, PJ, Shin, MS, Lee, JH et al. (2001) Nuclear localization of β-catenin is an important prognostic factor in hepatoblastoma. J. Pathol. 193, 483–490) (Lopez-Terrada, D., Gunaratne, PH, Adesina, AM, Pulliam, J., Hoang, DM, Nguyen, Y. et al. (2009) Histologic subtypes of hepatoblastoma are characterized by differential canonical Wntand Notch pathway activation in DLK+ precursors. Hum. Pathol. 40, 783–794).Furthermore, the increase in nuclear beta-catenin was positively correlated with the increase in cyclin D1 positive staining. Cyclin D1 is a nuclear factor protein that controls cell proliferation (Takayasu, H., Horie, H., Hiyama, E., Matsunaga, T., Hayashi, Y., Watanabe, Y. et al. (2001) Frequent deletions and mutations of the β-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma. Clin. Cancer Res. 7, 901–908). Therefore, the development of lead compounds, beta-catenin inhibitors, for the treatment of cancers such as hepatoblastoma is of great significance.

[0004] Due to the long development cycle of traditional drug development, drug repurposing (repurposing existing drugs) has become an effective strategy in drug development. New drug development is a process with a high failure rate, high cost, and slow pace, while drug repurposing offers advantages such as lower development costs and shorter development times. Therefore, reusing existing drugs to treat common and rare diseases is becoming increasingly attractive. Although various data and experimental methods exist to aid in screening candidates for drug repurposing, the key technical approaches and challenges still need to be addressed.

[0005] This invention utilizes structure-based virtual screening of a library of FDA-approved drug compounds to identify inhibitors that effectively dock with beta-catenin proteins. By combining virtual screening with molecular docking techniques, it discovers lead compounds for hepatoblastoma inhibitors, which is of great significance for drug repositioning. Summary of the Invention

[0006] The purpose of this invention is to provide a protein-based drug repositioning method and its application.

[0007] The design concept of this invention is as follows: First, the three-dimensional structure PDB file of beta-catenin protein is obtained from the AlphaFold database, and the receptor protein structure is optimized. Then, an FDA-approved drug small molecule compound and the beta-catenin co-crystallized ligand inhibitor RS6452 are used as a ligand library. The docking center coordinates are determined by the position of the ligand inhibitor RS6452 in the beta-catenin co-crystallized complex. Virtual screening is performed using Sailvina software, and the binding energy scores of the docking results are sorted. Finally, Autodock software is used to perform molecular docking between the virtual screening results and the beta-catenin protein to screen out the optimal binding conformation.

[0008] Based on the above design concept, the present invention adopts the following technical solution: A protein-based drug repositioning method and its application, characterized by comprising the following steps: (1) Preparation of receptor protein: Obtain the three-dimensional structure pdb file of beta-catenin protein from the AlphaFold database, optimize the receptor protein structure according to the protonation / charge distribution, and complete the receptor preparation work; (2) Preparation of ligand library: Download FDA-approved drug small molecule compounds from the Pubchem database as ligand small molecule library, and include the beta-catenin co-crystallized ligand inhibitor RS6452 as a reference standard for evaluating the screened ligands into the ligand library to complete the ligand preparation work. (3) Setting up the docking active pocket: Determine the docking center coordinates based on the position of the ligand inhibitor RS6452 in the beta-catenin co-crystallized complex (PDB ID: 7ZRB), and set the docking radius; (4) Virtual screening of compounds: Set the number of docking times in the virtual screening software, dock the compounds to be screened into the receptor protein activity pocket, sort the docking binding energy scores, and obtain candidate compounds with potential inhibitory activity against beta-catenin protein. (5) Molecular docking: Molecular docking software is used to perform molecular docking with beta-catenin protein on the selected ligand small molecules with high docking binding energy scores to obtain the conformation with the best docking effect.

[0009] Furthermore, in step (1), the three-dimensional structure pdb file of beta-catenin protein obtained from the AlphaFold database is a protein compound with high resolution and complete amino acid peptides.

[0010] Furthermore, step (1) includes hydrogenation of the receptor protein, filling in missing residues, and structural repair of the side chains based on the protonation / charge distribution.

[0011] Furthermore, in step (2), since RS6452 is known to be an effective inhibitor of beta-catenin, it is selected as a reference standard for evaluating the screened ligands and included in the ligand library.

[0012] Further, in step (3), the three-dimensional structure of the beta-catenin and inhibitor co-crystallized complex (PDB ID: 7ZRB) is obtained from the RCSB Protein Data Bank database. The center coordinates of the active pocket are set as (13.817, -8.312, 11.485) based on the central site of the bound inhibitor, and the docking radius is set as 22.5 Å.

[0013] Furthermore, in step (4), the docking screening is performed virtually using the Autodock vina program in the Sailvina software. Specifically, the recipient protein folder is entered in the Sailvina software to generate a prepare.pdbqt file; the docking center coordinates and docking radius are entered, and a config.txt file is output to prepare the docking configuration; the ligand pdb file is entered and converted into pdbqt format using the Openbabel program in Sailvina. The output pH of the ligand small molecule is 7.4, and the energy is minimized using the MMFF94 force field; the docking number is set to one, the docking results are obtained, and the results are sorted according to the docking binding energy score.

[0014] Furthermore, in step (5), the molecular docking step specifically involves setting the same active site center coordinates and docking sphere radius as in step (3), designing docking active pockets, and selecting the conformation with the best docking effect based on the poses score value and combining them into a composite compound.

[0015] The innovation of this invention lies in obtaining the co-crystallized complex (PDB ID: 7ZRB) of beta-catenin and the inhibitor RS6452 from the RCSB Protein Data Bank database in step (2). Previous studies have found that in co-immunoprecipitation studies of HCT116 cells transfected with myc-labeled T-cytokine 4 (TCF-4), the inhibitor RS6452 eliminated the association between beta-catenin and TCF-4. Crystallographic analysis of the beta-catenin armadillo repeat domain structure showed that RS6452 and TCF-4 share a common binding site in the hotspot binding region near Lys508. These results highlight the potential of this novel beta-catenin inhibitor as an anticancer drug. Therefore, this invention selected the inhibitor RS6452 as a reference standard for evaluating the screened ligands and included them in the ligand library. Virtual screening of ligands with a binding energy lower than that of the inhibitor RS6452 may become potential inhibitors of beta-catenin protein. The introduction of this evaluation standard will effectively improve the accuracy of the research.

[0016] The beneficial effects of this invention lie in the promoting effect of drug repositioning on drug development. This invention can universally identify potential disease treatments, and is not limited to a specific disease(s) or drug(s). New drug development is a process with a high failure rate, high cost, and slow pace, while drug repositioning has the advantages of low development cost and short development time. Therefore, reusing existing drugs to treat common and rare diseases is becoming increasingly attractive. This invention establishes for the first time a drug repositioning method for anti-hepatoblastoma drugs targeting beta-catenin. This method can obtain clues about active compounds in a short time, focusing the research target from tens of thousands of compounds to dozens, greatly improving the speed and efficiency of compound screening and shortening the cycle of new drug research. Attached Figure Description

[0017] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is the crystal structure of the beta-catenin protein of the present invention; Figure 2 This is the binding site for the receptor protein and the ligand small molecule of the present invention; Figure 3 This is a schematic diagram illustrating the molecular docking analysis of the receptor protein and the compound of this invention. Detailed Implementation

[0018] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them, and should not be construed as limiting the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Example

[0019] A protein-based drug relocation method and its application.

[0020] The specific steps are as follows: Step (1) Preparation of receptor proteins, specifically including: ① Obtain the 3D structure PDB file of the human beta-catenin protein (ID: AF-P35222-F1) from the AlphaFold database. The protein crystal structure is shown below. Figure 1 As shown.

[0021] ② Perform structural repair on the protein, specifically including hydrogenation, completion of missing residues and side chains, and protonation of polar amino acids, and save it in the receptor protein folder.

[0022] Step (2) Preparation of the ligand library, specifically including: Download the SDF format files of 2347 FDA-approved small molecule drug compounds (L4200, TargetMol) from the Pubchem database, and use OpenBabel software (http: / / openbabel.org / wiki / Main_Page) to batch convert the SDF format files to PDB format files and save them in the ligand small molecule folder.

[0023] Step (3) determines the active pocket for virtual screening and molecular docking, and the specific steps include: ① Determination of the docking center site: The center coordinates of docking were determined using the center-on-ligand method. The co-crystallized crystal structure PDB file of the beta-catenin protein (PDB ID: 7ZRB) was downloaded from the PDB database. The receptor protein was processed using Pymol software to remove water molecules and redundant sequences, and the central carbon atom of the co-crystallized ligand inhibitor RS6452 was located. The PDB file of the complex was opened using Notepad++ software, and the coordinates of the central carbon atom of the co-crystallized ligand (13.817, -8.312, 11.485) were found. These coordinates are the center coordinates of the docking pocket required for subsequent virtual screening and molecular docking. The binding site is as follows: Figure 2 As shown.

[0024] ② Setting the radius of the active pocket: Set the radius of the active pocket to 22.5 Å.

[0025] Step (4) Use Sailvina software to perform the first virtual screening, including the following steps: ① In the Sailvina software, input the folder containing the receptor and output a receptor protein file named prepare.pdbqt. Determine the center coordinates of the docking using the center-on-ligand method. Input the site where beta-catenin directly interacts with the co-crystallized ligand inhibitor RS6452 (13.817, -8.312, 11.485) into the docking active site. The binding site is as follows: Figure 2 As shown, the radius parameter of the docking sphere is set to 22.5Å, and a docking configuration file named config.txt is output.

[0026] ② Input the ligand pdb folder into the Sailvina software and convert it to pdbqt format using Openbabel software. The output pH is 7.4. The MMFF94 force field is used for energy minimization. Save the obtained ligand file in the ligand small molecule folder. Set the docking number to one and obtain the docking results. Sort them according to the docking binding energy score.

[0027] Table 1 shows the top ten compounds selected.

[0028] Table 1 Top Ten Compounds Screened Radotinib 926037-48-1 -7.8 Pimozide 2062-78-4 -7.7 Conivaptan 210101-16-9 -7.5 Lumacaftor 936727-05-8 -7.5 Capmatinib 1029712-80-8 -7.5 Larotrectinib 1223403-58-4 -7.5 Lifitegrast 1025967-78-5 -7.4 Eltrombopag 496775-61-2 -7.4 Fluorescein 2321-07-5 -7.4 Entrectinib 1108743-60-7 -7.4 Step (5) Use Autodock software to perform molecular docking between the top four compounds selected in step (4) and the beta-catenin protein. Select the conformation with the best docking effect based on the poses score, including the following steps: ① Import the beta-catenin protein into the macromolecular protein window, and use the Autodock tools module to perform hydrogenation on the receptor protein and calculate the Gasteiger charge. ② Import the compounds screened in step (4) into the small molecule ligand window in batches, use the Autodock tools module to perform hydrogenation calculation of the ligands Gasteiger charge processing, and save it as pdbqt format; ③ Docking box preparation: Use Notepad++ software to find the center coordinates of the original ligand (13.817, -8.312, 11.485) in the txt file of the original co-crystallized ligand complex, set it as the docking site in Autodock vina, use Grid--Grid BOX to set the docking parameters, the box radius is 22.5 Å, and save the relevant parameters in a file in gpf format.

[0029] ④ Molecular docking: Run the Grid module to generate a map format file, select semi-flexible docking, set the docking count to 50, and output a DPF file. Run Autodock to generate the corresponding DLG file and view the docking results.

[0030] This invention selected four small molecule compounds based on the scoring results. The docking analysis diagram is shown below. Figure 3 As shown in Table 2, the docking binding energy scores are as follows.

[0031] Table 2. Binding Energy Scoring Table for Four Compounds

[0032] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0033] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A protein-based drug repositioning method characterized in that, Includes the following steps: (1) Preparation of receptor protein beta-catenin: Obtain the three-dimensional structure pdb file of beta-catenin protein from the AlphaFold database and optimize the receptor protein structure; (2) Preparation of ligand molecule library: Download FDA-approved drug small molecule compounds from Pubchem database as ligand small molecule library, and include beta-catenin co-crystallized ligand inhibitor RS6452 as a reference standard for evaluating the screened ligands in the ligand library. Use Openbabel software to convert the ligands into pdbqt format files. (3) Setting up the docking active pocket: Determine the docking center coordinates based on the position of the ligand inhibitor RS6452 in the beta-catenin co-crystallized complex, and set the docking radius; (4) Virtual screening of compounds: Set the number of docking times in the virtual screening software, dock the compounds to be screened into the receptor protein activity pocket, sort the docking binding energy scores, and obtain candidate compounds with potential inhibitory activity against beta-catenin protein. (5) Molecular docking: Molecular docking software is used to perform molecular docking with beta-catenin protein on the selected ligand small molecules with high docking binding energy scores to obtain the conformation with the best docking effect.

2. The drug repositioning method according to claim 1, characterized in that, In step (1), the three-dimensional structure of beta-catenin protein is obtained from the AlphaFold database, and the beta-catenin receptor protein is subjected to structural repair treatment to complete the missing residues and side chains.

3. The drug repositioning method according to claim 1, characterized in that, In step (2), the compounds to be screened are selected from the FDA-approved drug compound library in the Pubchem compound library. The small molecule RS6452, a co-crystallized ligand inhibitor of beta-catenin protein, is included in the ligand library as a reference standard for evaluating the screened ligands. The ligands are converted into pdbqt format files using Openbabel software.

4. The drug repositioning method according to claim 1, characterized in that, In step (3), the center coordinates of docking are determined using the center on ligand method. The center coordinates of docking are determined by the position of the ligand inhibitor RS6452 in the beta-catenin co-crystallized complex, and the docking radius is set to 22.5 Å.

5. The drug repositioning method according to claim 1, characterized in that, The virtual screening in step (4) uses Sailvina software. The receptor protein file, docking center coordinates and docking active pocket radius, and ligand library file are input. The number of docking times is set to perform structure-based virtual screening, and the obtained docking binding energy scores are sorted.

6. The drug repositioning method according to claim 1, characterized in that, In step (5), the Autodock software is used to perform molecular docking of the top four compounds in the virtual screening with the beta-catenin protein. The conformation with the best docking effect is selected based on the poses score obtained from the docking and a complex file is generated.

7. The drug repositioning method according to claim 6, wherein the first four compounds include Radotinib as shown in Formula 1, Pimozide as shown in Formula 2, Conivaptan as shown in Formula 3, and Lumacaftor as shown in Formula 4. 。

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