WO2021077520A1

WO2021077520A1 - Application of virtual screening in the preparation of protein kinase inhibitor and drug lead compound

Info

Publication number: WO2021077520A1
Application number: PCT/CN2019/119887
Authority: WO
Inventors: 张娜; 李春琼; 张雪文; 孙国辉; 赵丽娇; 钟儒刚
Original assignee: 北京工业大学
Priority date: 2019-10-21
Filing date: 2019-11-21
Publication date: 2021-04-29
Also published as: CN110718268A; CN110718268B

Abstract

The invention belongs to the field of pharmaceutical chemistry, and provides an application of virtual screening in the preparation of a protein kinase inhibitor and a drug lead compound, said screening method comprising the following steps: (1) obtaining a candidate compound by means of virtual screening; (2) testing the in vitro protein kinase activity and anti-tumor cell proliferation activity of the candidate compound obtained in step (1), and screening to obtain a target compound. The screening method obtains a target compound which has inhibitory activity on CK2 and lung cancer cells A549, and the method has such advantages as being time-saving, effort-saving, cost-saving, and high-efficiency.

Description

Application of virtual screening in the preparation of protein kinase inhibitors and drug lead compounds

Technical field

The invention relates to the field of medicinal chemistry, in particular to a method for screening protein kinase inhibitors and the application of drug lead compounds in protein kinase inhibitors or/and anti-tumor proliferation drugs.

Background technique

Protein Kinase CK2 (Protein Kinase CK2) is a highly conserved serine/threonine protein kinase with diverse biological properties of substrate proteins. It can catalyze more than 300 protein substrates and participate in a variety of cellular regulatory processes, including Cell growth and proliferation, cell survival and apoptosis, embryonic development and organ formation, and transcriptional regulation are key regulators of many cellular processes. Studies have found that when cells are stimulated by certain conditions that lead to a forced down-regulation of CK2 levels in the nucleus, even a moderate down-regulation can lead to large-scale cell death through the apoptotic mechanism. Therefore, CK2 has become an important anti-cancer therapeutic target.

At present, although most CK2 inhibitors are ATP-competitive inhibitors, the high degree of conservation of ATP active sites in the protein kinase family makes this type of inhibitors challenging in terms of their selectivity. For example: entering clinical stage Ⅱ CX-4945, while described as having a high selectivity, but also inhibit other kinases with at least twelve nanometers IC ₅₀ values, and the comparison and that inhibits CK2, it is more effective Clk2 . Therefore, we hope to research and develop high-affinity allosteric inhibitors of CK2 by targeting non-ATP sites.

With the vigorous development of computer-aided drug design, virtual drug screening has effectively improved the high-cost and high-risk problems in the traditional drug development process. It has become a complementary technology to high-throughput screening and is the discovery of lead compounds and their activity optimization. Important method. Therefore, the establishment of a fast and efficient virtual screening method for protein kinase inhibitors is of great significance for the discovery of new protein kinase allosteric inhibitors and their application in anti-tumor proliferation.

Summary of the invention

The purpose of the present invention is to provide a rapid and efficient method for screening protein kinase inhibitors and the application of target compounds in protein kinase inhibitors or/and anti-tumor drugs.

The present invention adopts the following technical solutions:

A method for screening protein kinase inhibitors includes the following steps:

(1) Obtain candidate compounds through virtual screening methods;

(2) The candidate compound obtained in step (1) is screened for biological activity to obtain the target compound.

According to the present invention, the virtual screening in step (1) is preferably a virtual screening method based on a combination of a pharmacophore model and molecular docking;

Preferably, the pharmacophore model construction method uses Discovery Studio 4.0 software, and the molecular docking method uses GOLD software.

According to the present invention, the biological activity screening in step (2) is a kinase activity detection experiment and an anti-tumor cell proliferation experiment;

Preferably, the kinase activity detection experiment method is the ADP-Glo kinase activity detection method, and the anti-tumor cell proliferation experiment method adopts the Cell Counting Kit-8 (CCK-8) method.

As an implementation method, the screening method of the present invention includes the following steps:

(S1.1) Obtain the three-dimensional structure of the co-crystal of protein kinase CK2 and inhibitor from the protein data bank, and reserve and set the active pocket according to the bound inhibitor;

(S1.2) Use the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal obtained in step S1.1 to construct a pharmacophore model based on the receptor protein kinase CK2, and use this pharmacophore model as a query formula for small molecules Preliminary screening of the compound library based on the matching degree value to obtain the preliminary screening compound library;

(S1.3) According to the active pocket set in step (S1.1), molecular docking software is used for molecular docking with the compounds screened in step S1.2 and scoring and sorting, further screening to obtain a refined compound library, and passing the evaluation Candidate compounds are obtained by docking the interaction mode of the molecule and CK2;

(S2) Use the candidate compound obtained in step S1.3 to perform a kinase activity detection experiment and an anti-tumor cell proliferation experiment to obtain the target compound.

According to the screening method of the present invention, in step S1.1, the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal is the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal in the existing protein database, or the protein kinase CK2 and other inhibitors Eutectic three-dimensional structure;

According to the screening method of the present invention, in step S1.2, the pharmacophore model construction method is: using Discovery Studio 4.0 to construct a receptor-based pharmacophore model, using molecular substances that have been determined to be active and inactive to the receptor The screen library module is used for model verification. The obtained pharmacophore model contains 3-5 effective pharmacodynamic characteristic elements, and the matching degree value is greater than 2.5-3.5 to screen the candidate small molecule compound library, and obtain Preliminary screening of compound libraries.

Preferably, the small molecule compound library in step S1.2 is a ChemBridge fragment library.

According to the screening method of the present invention, in step S1.3, the molecular docking program is preferably GOLD, and other molecular docking software can be used instead, for example: one or more of AutoDock vina, FlexX or Glide;

Preferably, when using GOLD for docking scoring, the Goldscore scoring function is used;

Preferably, the refined compound library is preferably the compounds ranked in the top 20-100, and the candidate compounds are preferably 6-10 compounds that have a similar mode of action as the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal described in step S1.1 compound of.

The present invention further provides a target compound obtained by the screening method for protein kinase inhibitors for use in the preparation of protein kinase inhibitors or/and anti-tumor drugs. The target compound is a compound having a structure represented by formula I and formula II or its pharmaceutically Acceptable salt:

Use of the compounds represented by formula I and formula II or their pharmaceutically acceptable salts as inhibitors of protein kinase CK2.

Furthermore, the compounds represented by formula I and formula II have a certain inhibitory effect on protein kinase CK2. When the compound concentration is 64 μM, the inhibition rate of protein kinase CK2 is 60% and 26%, respectively; when the compound concentration is 256 μM, it has inhibitory effect on protein kinase CK2. The inhibition rates are 72% and 40% respectively;

The use of the compounds represented by formula I and formula II or their pharmaceutically acceptable salts as anti-tumor drugs.

Furthermore, the compounds represented by formula I and formula II have certain anti-tumor cell proliferation activity. The tumor cell line is human lung cancer A549, and its IC ₅₀ values are 23.08 μM and 8.81 μM, respectively.

The protein kinase inhibitor screening method and its target compound established in the present invention have the following advantages: 1) Through the virtual screening method based on pharmacophore model and molecular docking, it saves time and cost, improves screening efficiency and compound hits 2) In vitro kinase activity detection experiments show that the screened target compounds I and II have a significant inhibitory effect on protein kinases, and have application prospects in the prevention or/and treatment of protein kinase-mediated diseases; 3) Anti-tumor cell proliferation The experimental results show that the target compounds I and II obtained by screening have obvious anti-proliferative effects on human lung cancer cells and have potential clinical treatment prospects.

Description of the drawings

Figure 1 is a diagram of the constructed receptor-based pharmacophore model;

Table 1 shows the structural formulas of candidate compounds I to VI and their inhibitory rates on protein kinase CK2 activity at concentrations of 64 μM and 256 μM;

Figure 2 shows the proliferation inhibition rate of target compounds I and II on human lung cancer cells.

Detailed ways

The present invention will be further described below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

Example 1:

The virtual screening steps are as follows:

1) Obtain the three-dimensional structure of the co-crystal of protein kinase CK2 and inhibitor from the protein data bank (protein kinase CK2 search number: 5OTZ).

2) Log in to the ChemBridge official website (https://www.chembridge.com/), find the Fragment library, download all the available fragment compounds, and get a database containing 13,802 small molecule compounds. And the passed Build 3D Database function module is added to Discovery studio 4.0 (the following steps 3-7 are all completed in Discovery studio 4.0) to form a small molecule compound library that can be used for subsequent screening.

3) Use the Prepare Protein module to process the protein file of the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal CK2 downloaded in step 1) to remove multiple protein conformations, supplement incomplete amino acid residues, and hydrogenate proteins, etc. .

4) Define CK2 in the co-crystal complex of protein kinase CK2 and inhibitor as the receptor molecule, use the From Receptor Cavities function module to find the active site, and then retain the active site where the inhibitor is located (here refers to the protein kinase CK2 reported in the literature) ΑD pocket), delete other active sites.

5) According to the above selected binding site, use Edit and Cluter Pharmacophore features under Pharmacophore module to generate an interaction model, which contains 116 hydrogen bond acceptor features, 209 hydrogen bond donor features, and 110 hydrophobic features for a total of 435 Characteristic elements. And through automatic clustering analysis combined with the method of manually selecting the characteristic elements that interact with important residues at the active site, the screen contains 5 hydrogen bond acceptor features, 7 hydrogen bond donor features and 6 hydrophobic features. Preliminary pharmacophore model (with excluded volume).

6) Select 10 molecules that have affinity for the αD pocket of CK2 and 8 molecules that are inactive to CK2 from the reported literature of protein kinase CK2 inhibitors to form a test set, and use the Screen Library module to screen and verify 18 characteristic elements. In order to obtain the key pharmacodynamic characteristic elements, a pharmacophore model containing 3-5 characteristics is finally obtained. For example, when the 5OTZ protein is used as the acceptor molecule, the characteristics of two hydrogen bond donors are obtained as shown in Figure 1. Pharmacophore model with hydrophobic characteristics.

7) Using the Search 3D Database module, using the above-mentioned pharmacophore model containing the four characteristic elements as a query formula, the small molecule compound library in step 2) is matched and screened to obtain a preliminary screening database containing 92 fragment compounds.

8) According to the active sites established in step 4), use GOLD software to perform molecular docking on the 92 compounds in the preliminary screening database, and sort the 92 compounds from high to low according to the docking scoring results, and screen out the top 20 with a score The compounds constitute a fine screening compound library.

9) Further analyze the interaction mode of the above 20 compounds with active pocket residues. Finally, 6 candidate compounds (compounds I to VI) were screened out. These candidate compounds can form polar interactions with the backbone carbonyl groups of Pro159 and Val162, and can form hydrophobic interactions with the hydrophobic pockets of Try136, Ile133, Met221 and Met225. This is related to the interaction mode between inhibitors in PDB ID 5OTZ and protein kinases. similar.

The biological activity screening steps are as follows:

1) The ADP-Glo kinase activity detection method is used to determine the in vitro activity of protein kinase CK2: 5μl of different concentrations of the test compound (inhibitor), 10μl of protein kinase CK2 solution and 10μl of substrate/ATP are added to a 96-well plate with a white background. The solution yielded a total volume of 25 μl of the reactant. The reaction was started by adding the substrate/ATP to the kinase and incubated at room temperature for 60 min. Then add 25μl of ADP-Glo reagent to each reaction well to stop the reaction and consume the remaining ATP in each well, and incubate at room temperature for 40min. Finally, add 50μl kinase detection reagent to each well, incubate at room temperature for 30min to convert the generated ADP into ATP and react with luciferase in the detection reagent to emit light, and then detect its luminescence value with a microplate reader. The inhibitory effects of different compounds on protein kinase CK2 at different concentrations were calculated by comparing the 100% inhibition of the reaction mixture with no enzyme (negative control) and the 0% inhibition of the reaction mixture with 5% DMSO (positive control).

According to the above experimental method, the inhibitory effect of virtual screening candidate compounds on protein kinase CK2 was determined. The results are shown in Table 1. Candidate compounds I and II have good inhibitory effects on protein kinase CK2 in vitro, and the compound concentration is 64μM. The inhibition rates of CK2 were 60% and 26%, respectively; when the compound concentration was 256 μM, the inhibition rates of protein kinase CK2 were 72% and 40%, respectively.

2) Anti-tumor proliferation determination of compounds with high kinase activity inhibitors: The CCK-8 method was used for the experimental determination. Spread 2×10 ⁴ tumor cells in logarithmic growth phase on a 96-well plate, _{incubate in a 5% CO 2} , 37°C incubator for 24 hours until the cells adhere to the wall, add 100 μl inhibitors of different concentrations to each well, and incubate for 48 hours Then add 10μl of CCK-8 reagent, and measure its absorbance at 450nm with a microplate reader after 2h. Calculate the inhibition rate, make a nonlinear regression curve, and get the IC ₅₀ value.

As shown in Figure 3, compounds I and II showed significant inhibitory activity on human lung cancer cells, with IC ₅₀ values of 23.08 μM and 8.81 μM, respectively.

In summary, the method for screening protein kinase inhibitors provided by the present invention can quickly and efficiently screen out lead compounds of protein kinase CK2 inhibitors from a large number of compounds, and provide theoretical basis and experimental guidance for further structural optimization.

The above are the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention are all included in the protection scope of the present invention.

Claims

A method for screening protein kinase inhibitors, which is characterized in that it comprises the following steps:

(1) Obtain candidate compounds through virtual screening methods;

(2) The candidate compound obtained in step (1) is screened for biological activity to obtain the target compound;

The virtual screening described in step (1) is a virtual screening method based on the combination of pharmacophore model and molecular docking;

The biological activity screening described in step (2) is a kinase activity detection experiment and an anti-tumor cell proliferation experiment.
A method for screening protein kinase inhibitors according to claim 1, wherein the method for constructing a pharmacophore model in step (1) adopts Discovery Studio 4.0 software, and the method for molecular docking adopts GOLD software.
A method for screening protein kinase inhibitors according to claim 1, wherein step (2) the kinase activity detection method is the ADP-Glo kinase activity detection method, and the anti-tumor cell proliferation experiment method uses Cell Counting Kit- 8 (CCK-8) method.
A method for screening protein kinase inhibitors according to claim 1, characterized in that it specifically comprises the following steps:

S1.1. Obtain the three-dimensional structure of the co-crystal of protein kinase CK2 and inhibitor from the protein data bank, and reserve and set the active pocket according to the bound inhibitor;

S1.2. Use the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal obtained in step S1.1 to construct a pharmacophore model based on the receptor protein kinase CK2, and use this pharmacophore model as a query formula for small molecule compounds Perform preliminary screening of the library based on the matching degree value to obtain a preliminary screening compound library;

S1.3. According to the active pocket set in step S1.1, molecular docking software is used to do molecular docking with the compounds screened in step S1.2, and then they are scored and sorted. The refined compound library is obtained by further screening, and the docking molecule and the compound are evaluated. CK2 interaction mode to obtain candidate compounds;

S2. Use the candidate compound obtained in step S1.3 to perform a kinase activity detection experiment and an anti-tumor cell proliferation experiment to obtain the target compound.
The method according to claim 4, wherein in step S1.1, the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal is the three-dimensional structure of the protein kinase CK2 and the inhibitor co-crystal in the existing protein database, or the protein The three-dimensional structure of kinase CK2 co-crystal with other inhibitors;

In step S1.2, the method of constructing the pharmacophore model is: using Discovery Studio 4.0 to construct a receptor-based pharmacophore model, using molecular substances that have been determined to be active and inactive to the receptor, and using Screen The Library module performs model verification, and the obtained pharmacophore model contains 3-5 effective pharmacodynamic characteristic elements, and uses a matching degree value greater than 2.5-3.5 to screen small molecule compound libraries to obtain a preliminary screening compound library;

The small molecule compound library in step S1.2 is a ChemBridge fragment library;

In step S1.3, the molecular docking program is preferably GOLD, or other molecular docking software may be used instead, for example: one or more of AutoDockvina, FlexX or Glide;

When using GOLD for docking scoring, use the Goldscore scoring function;

The refined compound library is preferably the compounds ranked in the top 20-100, and the candidate compounds are preferably 6-10 compounds that have a similar mode of action to the three-dimensional structure of the protein kinase CK2 and inhibitor co-crystal described in step S1.1.
The target compound obtained by the screening method of claims 1-5 is a compound having the structure shown in Formula I and Formula II or a pharmaceutically acceptable salt thereof:
The use of the compound with the structure shown in formula I and formula II according to claim 6 or a pharmaceutically acceptable salt thereof in the preparation of protein kinase CK2 inhibitors or/and anti-tumor drugs.
An application of a compound represented by formula I and formula II or a pharmaceutically acceptable salt thereof in the preparation of a protein kinase CK2 inhibitor and/or an antitumor drug;
The use of the compound of claim 8 or a pharmaceutically acceptable salt thereof in the preparation of protein kinase CK2 inhibitors and/or anti-tumor drugs, characterized in that the compounds represented by formula I and formula II have certain protein kinase CK2 inhibition Effect, when the compound concentration is 64μM, the inhibition rate of protein kinase CK2 is 60% and 26%, respectively; when the compound concentration is 256μM, the inhibition rate of protein kinase CK2 is 72% and 40%, respectively;

The compounds represented by formula I and formula II have certain anti-tumor cell proliferation activity. The tumor cell line is human lung cancer A549, and its IC 50 values are 23.08 μM and 8.81 μM, respectively.