Report on Reverse Targeting of Natural Small Molecular Compounds

1 Purpose

Carry out reverse virtual screening for one compound provided by the customer, and return the results to the customer.

2 Materials and Methods

2.1 Reverse virtual screening materials

One compound provided by the customer is shown in Table 1.

Table 1. Five compounds provided by the customer

one

2.2 Reverse Virtual Filtering Process

Docking software: Glide or Vina software.

Methods: The receptor based reverse virtual screening method was based on the principle of molecular docking. Small molecular compounds were received one by one to the target active sites in the target database. The compounds were ranked according to the binding energy of the compounds and targets. The lower the binding energy, the higher the ranking, and the greater the possibility of potential targets.

Protein docking screening library: We use docking software to perform reverse virtual docking screening based on the Scpdb database and our self-developed reverse virtual screening database. The protein database has a total of 23456 kinds of proteins. The protein database integrates all known crystal protein structure type data in the current RCSB PDB database, and has the most comprehensive protein structure data information. Submit the reverse virtual screening task for one compound and return the docking results of the first 500 molecules.

3 Results and Analysis

Each compound result file includes the top 500 docking file information, and the top 500 docking results are shown in the list top_ Target_ 500.csv file, we selected the top 50 small molecules for focus analysis, as shown in the following table:

The small molecular phases with the best energy and the most clusters were selected for the result analysis.

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Taking the target result ranking first in the reverse virtual filtering of the compound1 Whole database as an example, this paper briefly introduces how to view the reverse virtual filtering result through target information analysis and combined with pattern analysis. The first target of compound1 is Cytochrome P450 11B2, mitochondrial [Homo sapiens] (the first column), and the target protein file is 4dvq_ 1CA_ 10_ [J] (The third column means PDBID: 4dvq, crystal structure ligand name 1CA, chain ID: J chain). During screening, if the screened target protein is human protein and the non screened protein is from other species, it is necessary to consider the sequence homology between the protein from other species and human protein, otherwise the target can be abandoned directly.

3.2.1 Target information analysis

The first target of Compound1 is cyclin dependent kinase 2 isoform 1 [Homo sapiens], and the target related information is in the Top_ Target_ 500.csv file, refer to the help document for details http://reversedock.vslead.com/index.php?r=site/help 。 Based on the understanding of the pharmacological activities and therapeutic fields of known compounds_ Target_ Target UNP in 500.csv file_ AC (fourth column) data, corresponding to the UniProt database, search for more comprehensive target information, and finally determine the potential target of the compound.

3.2.2 Analysis of the binding mode of compounds and target proteins

The first result file of compound1 is 4dvq_ 10_ 1CA. Pdbqt and 4dvq_ 10_ 1CA.log。 4dvq_ 10_ 1CA.log is the conformation score of the compound after docking. Analyze 4dvq_ 10_ 1CA. The conformation of the pdbqt compound after docking can be opened with visualization software (such as Pymol, if Pymol is opened with problems, the compound bond can be displayed with problems, and Discovery Studio can be used) to combine with the three-dimensional structure of the protein (here PDBID: 4dvq, in the http://www.rcsb.org/ Download it), analyze the compound protein binding mode (Figure 2) and electrostatic surface binding (Figure 3)

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Fig. 2 Binding mode diagram A) Crystal structure pdb: 4dvq binding mode diagram; B) Combination mode diagram of compound1 and pdb: 4dvq

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Fig. 3 Electrostatic surface diagram Crystal junction PDB: 4DVQ, rod compounds are ligands 1CA (green) and COM1 (carton) respectively.

This part is the most extensive business to be done by customers after the end of the preliminary data. It is provided here for reference only

4 Drug Molecular Design and Chemical Modification Based on Identification Protein

Computer aided drug design

Based on the identification of the three-dimensional structure of the obtained protein, the physical and chemical properties around the XYZ protein binding pocket were thoroughly analyzed. The report is as follows: (a) Before mutation, small molecules surrounded hydrophobic pockets, and hydrophilic amino acids R125 and E154 formed hydrogen bond rivets with them, making them firmly fixed in the albumen; (a) After mutation: the hydrophobic environment is roughly unchanged, but the amino acids N125 and I154 cause large gaps, and the hydrophilicity is converted into hydrophobic pockets, reducing the stability.

Molecular dynamics method

The interaction between XYZ protein and small molecule Hormone after mutation was calculated and analyzed by molecular dynamics method, in order to obtain the binding information of small molecule after mutation.

(a) After mutation, the region A of XYZ protein has no change, so no modification is made to this part;

(b) In Region B, there is a huge hole below the small molecule, which needs to be filled;

(c) A huge air attack in Region B, when small molecules combine stably, a "water hydrogen bond network system" consisting of 10-13 water molecules appears below;

(d) In the "water hydrogen bond network system", the energy (sphere size) and occupancy (sphere color) of water molecules can express the physical and chemical properties of the local region of the protein.

The specific summary is as follows: Most water molecules in Region B have very low energy and occupancy, indicating that the region is extremely hydrophobic; However, W1 water molecule has good energy and high occupancy, indicating that W1 water molecule is extremely stable, and the surrounding amino acids are extremely hydrophilic amino acids, which can be chemically modified by replacing W1 water molecules or by bridging

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Drug Design Based on Hormone

Chemical Fragment Growth Design

Based on the molecular dynamics simulation results and the relevant information of W1 water molecules, the chemical fragment structure of Region B was designed. The first chemical fragment designed will be able to effectively reach the vicinity of W1 water molecule, or replace it. A total of 50 parent core structures were designed, and all of them have the possibility of realizing organic synthesis experimental methods.

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Determination of the optimum chemical parent nucleus structure by molecular docking method

Using the molecular docking method, 50 mother nucleus structures were docked into the mutant protein XYZ structure respectively. Using the molecular docking weight value, it was determined that 3 small molecule drugs occupied a good weight position in the docking result. C2, C1 and C3 are used for the later chemical structure transformation.

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New parent nucleus structure modification and molecular docking

The chemical fragment structure modification of C2~3 was carried out. Especially, the second class C2 compound is the key research object. Based on C2 compound, 78 chemical fragments were designed, and then the 78 new chemical molecules were subject to Molecular Docking operation to predict their structural properties.

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Through molecular dynamics simulation, calculate the energy system between protein Protein and small molecule Ligand, rank the most energetic system, and get the top 50 small molecules with better energy system.

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chemical synthesis

After the compound structure is obtained, chemical synthesis is carried out.

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Cell activity test in vitro

(1) Tumor cell culture

(2) Cell activity test

Cytotoxicity, cell migration, live cell imaging, real-time dynamic live cell imaging, and non labeled live cell imaging.

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The very good result is that the activity experiment is good, especially the cell activity data IC50=45nm of 10 compounds such as C2-1 on cancer cells, which has a high potential for drug preparation.

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Construction of animal tumor model

Establish a biological activity laboratory to speed up animal experiments in vivo.

Breast cancer tumor cell lines were diluted and injected into mice, rats, rabbits and chimpanzees, and animal tumor models were successfully obtained.

Animal activity test in vivo

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