CN110718268A - Application of virtual screening in preparation of protein kinase inhibitor and drug lead compound - Google Patents
Application of virtual screening in preparation of protein kinase inhibitor and drug lead compound Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutical chemistry, and provides application of virtual screening in preparation of a protein kinase inhibitor and a drug lead compound, wherein the screening method comprises the following steps: (1) obtaining a candidate compound through virtual screening; (2) and (2) carrying out in-vitro protein kinase activity and anti-tumor cell proliferation activity detection on the candidate compound obtained in the step (1), and screening to obtain a target compound. The screening method of the invention obtains the target compound with inhibitory activity on CK2 and lung cancer cell A549, and the method has the advantages of time and labor saving, cost saving, high efficiency and the like.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a screening method of a protein kinase inhibitor and application of a drug lead compound in the protein kinase inhibitor or/and an anti-tumor proliferation drug.
Background
Protein kinase CK2(Protein kinase CK2) is a highly conserved serine/threonine Protein kinase, has the biological characteristics of substrate Protein diversification, can catalyze more than 300 Protein substrates, participates in various cell regulation processes, including cell growth and proliferation, cell survival and apoptosis, embryonic development and organ formation, transcriptional regulation and the like, and is a key regulator of a plurality of cell processes. It has been found that when cells are stimulated by certain conditions to cause a forced down-regulation of CK2 levels in the nucleus, even modest down-regulation can lead to extensive cell death through apoptotic mechanisms. Therefore, CK2 has become an important anti-cancer therapeutic target.
Currently, although most CK2 inhibitors have been shown to be ATP competitive inhibitors, such inhibitors are challenging in their selectivity due to the high degree of conservation of the ATP active site in the protein kinase family. For example: CX-4945, which entered clinical phase II studies, although described as highly selective, also inhibits at least twelve other nano-ICs50A kinase of value, and is more effective against Clk2 than it is against CK 2. Therefore, we hope to develop CK2 allosteric inhibitors with high affinity by targeting non-ATP sites.
With the vigorous development of computer-aided drug design, virtual drug screening effectively solves the problems of high cost and high risk in the traditional drug research and development process, becomes a technology complementary with high-throughput screening, and is an important method for finding lead compounds and optimizing the activity thereof. Therefore, the method for virtually screening the protein kinase inhibitor with high speed and high efficiency is established, and has important significance for the discovery of the novel protein kinase allosteric inhibitor and the application of the novel protein kinase allosteric inhibitor in the aspect of tumor proliferation resistance.
Disclosure of Invention
The invention aims to provide a rapid and efficient screening method of a protein kinase inhibitor and application of a target compound in a protein kinase inhibitor or/and an anti-tumor drug.
The invention adopts the following technical scheme:
a method for screening a protein kinase inhibitor, comprising the steps of:
(1) obtaining a candidate compound by a virtual screening method;
(2) and (2) carrying out biological activity screening on the candidate compound obtained in the step (1) to obtain the target compound.
According to the invention, the virtual screening in step (1) is preferably a virtual screening method based on a pharmacophore model and molecular docking combination;
preferably, the pharmacophore model construction method adopts Discovery Studio 4.0 software, and the molecular docking method adopts GOLD software.
According to the invention, the biological activity screening in the step (2) is a kinase activity detection experiment and an anti-tumor cell proliferation experiment;
preferably, the kinase activity detection test method is an ADP-Glo kinase activity detection method, and the anti-tumor Cell proliferation test method adopts a Cell Counting Kit-8(CCK-8) method.
As an implementation method, the screening method comprises the following steps:
(S1.1) acquiring a three-dimensional structure of a Protein kinase CK2 and inhibitor eutectic crystal from the Protein data bank, and reserving and setting an active pocket according to a combined inhibitor;
(S1.2) constructing a pharmacophore model based on receptor protein kinase CK2 by using the three-dimensional structure of the protein kinase CK2 and inhibitor eutectic obtained in the step S1.1, and performing primary screening based on a matching value on a small molecule compound library by using the pharmacophore model as an inquiry formula to obtain a primary screened compound library;
(S1.3) according to the active pocket set in the step (S1.1), performing molecular docking and grading sequencing on the compound screened in the step (S1.2) by using molecular docking software, further screening to obtain a fine screening compound library, and evaluating an interaction mode of a docking molecule and CK2 to obtain a candidate compound;
(S2) using the candidate compound obtained in the step S1.3 to perform a kinase activity detection experiment and an anti-tumor cell proliferation experiment, and obtaining the target compound.
According to the screening method, in the step S1.1, the three-dimensional structure of the protein kinase CK2 and inhibitor eutectic is the three-dimensional structure of the protein kinase CK2 and inhibitor eutectic in the existing protein database, or the three-dimensional structure of the protein kinase CK2 and other inhibitors eutectic;
according to the screening method of the invention, in step S1.2, the pharmacophore model construction method comprises the following steps: constructing a pharmacophore model based on a receptor by using discovery studio 4.0, adopting determined molecular substances active and inactive to the receptor, performing model verification by using a Screen Library module, obtaining the pharmacophore model containing 3-5 effective pharmacodynamic characteristic elements, and screening an alternative small molecular compound Library by using a matching degree value of more than 2.5-3.5 to obtain a primary screening compound Library.
Preferably, the library of small molecule compounds in step S1.2 is a chemcridge fragment library.
According to the screening method of the present invention, in step S1.3, the molecular docking procedure is preferably GOLD, and other molecular docking software may be used instead, for example: one or more of AutoDock vina, FlexX, or Glide;
preferably, when using GOLD for butt-scoring, a Goldscore scoring function is used;
preferably, the fine screening compound library is preferably a compound with a score of 20-100, and the candidate compound is preferably 6-10 compounds with a similar action mode with the three-dimensional structure of the protein kinase CK2 and the inhibitor cocrystal described in step S1.1.
The invention further provides an application of a target compound obtained by the protein kinase inhibitor screening method in preparation of a protein kinase inhibitor or/and an antitumor drug, wherein the target compound is a compound with a structure shown in a formula I and a formula II or a pharmaceutically acceptable salt thereof:
the application of the compound shown in the formula I and the formula II or the pharmaceutically acceptable salt thereof as a protein kinase CK2 inhibitor.
Furthermore, the compounds shown in the formula I and the formula II have certain inhibition effect on protein kinase CK2, and the inhibition rates on protein kinase CK2 are respectively 60% and 26% when the concentration of the compounds is 64 mu M; the inhibition rate of the compound concentration of 256 mu M on the protein kinase CK2 is 72 percent and 40 percent respectively;
the compound shown in the formula I and the formula II or the pharmaceutically acceptable salt thereof is applied to the application as the antitumor drug.
Furthermore, the compounds shown in the formula I and the formula II have certain antitumor cell proliferation activity, and the tumor cell strain is human lung cancer A549 and IC thereof50The values are respectively: 23.08. mu.M and 8.81. mu.M.
The screening method of the protein kinase inhibitor and the target compound thereof have the following advantages: 1) By adopting the virtual screening method based on the combination of the pharmacophore model and the molecular docking, the time and the cost are saved, and the screening efficiency and the compound hit rate are improved; 2) in-vitro kinase activity detection experiments show that the screened target compounds I and II have obvious inhibition effect on protein kinase and have application prospect in preventing or/and treating protein kinase mediated diseases; 3) the result of an anti-tumor cell proliferation experiment shows that the screened target compounds I and II have obvious anti-proliferation effect on human lung cancer cells and have potential clinical treatment prospect.
Drawings
FIG. 1 is a diagram of a constructed receptor-based pharmacophore model;
FIG. 2 shows the structural formulas of candidate compounds I-VI and the inhibition rates of the protein kinase CK2 activity at concentrations of 64. mu.M and 256. mu.M;
FIG. 3 shows the proliferation inhibition rates of target compounds I and II on human lung cancer cells.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1:
the virtual screening steps are as follows:
1) the three-dimensional structure of Protein kinase CK2 eutectic with inhibitor was obtained from Protein data bank (Protein kinase CK2 search number: 5 OTZ).
2) The ChemBridge official network (https:// www.chembridge.com /), found Fragmentlibrary, and downloaded all commercially available fragment compounds to obtain a database containing 13802 small molecule compounds. And the obtained Build 3D Database functional module is added into Discovery studio 4.0 (the following steps 3-7 are all completed in Discovery studio 4.0), so as to form a small molecule compound library for subsequent screening.
3) And (2) carrying out Protein file processing on the Protein kinase CK2 downloaded in the step 1) and CK2 with a three-dimensional structure of inhibitor eutectic by adopting a Prepare Protein module to remove Protein multicoconformation, supplement non-complete amino acid residues, hydrogenate Protein and the like.
4) The CK2 in the eutectic compound of the protein kinase CK2 and the inhibitor is defined as a receptor molecule, a FromReceptor Cavities functional module is used for searching an active site, then the active site where the inhibitor is located (referred to as an alpha D pocket of the protein kinase CK2 reported in the literature) is reserved, and other active sites are deleted.
5) According to the selected binding sites, an interaction model is generated by using Edit and clutterpharmacophore features under a Pharmacophore module, and 435 feature elements comprising 116 hydrogen bond acceptor features, 209 hydrogen bond donor features and 110 hydrophobic features are obtained. And screening to obtain a preliminary pharmacophore model (containing exclusion volume) containing 5 hydrogen bond acceptor characteristics, 7 hydrogen bond donor characteristics and 6 hydrophobic characteristics by combining automatic clustering analysis and a method for manually selecting characteristic elements interacting with important residues at the active site.
6) Selecting 10 molecules with affinity to an alpha D pocket of CK2 and 8 molecules without activity to CK2 from reported protein kinase CK2 inhibitor documents to form a test set, screening and verifying 18 characteristic elements by using a Screen Library module to obtain key pharmacodynamic characteristic elements, and finally obtaining a pharmacophore model containing 3-5 characteristics, for example, obtaining the pharmacophore model containing two hydrogen bond donor characteristics and two hydrophobic characteristics as shown in figure 1 when 5OTZ protein is used as an acceptor molecule.
7) And (3) matching and screening the small molecular compound library in the step 2) by using a Search 3D Database module and taking the pharmacophore model containing the four characteristic elements as an inquiry formula to obtain a primary screening Database containing 92 fragment compounds.
8) Performing molecular docking on 92 compounds in the primary screening database by using GOLD software according to the active sites established in the step 4), sorting the 92 compounds from high to low according to docking and scoring results, and screening out compounds which are scored and ranked at the top 20 to form a fine screening compound library.
9) The interaction pattern of the 20 compounds with the active pocket residues was further analyzed. Finally, 6 candidate compounds (compounds I to VI) were screened. These candidate compounds can form polar interactions with the backbone carbonyl groups of Pro159 and Val162 and hydrophobic interactions with the hydrophobic pocket formed by Try136, Ile133, Met221 and Met225, similar to the interaction pattern of inhibitors and protein kinases in PDB ID 5 OTZ.
The biological activity screening steps are as follows:
1) the ADP-Glo kinase activity detection method is adopted to measure the in vitro activity of the protein kinase CK 2: mu.l of different concentrations of the test compound (inhibitor), 10. mu.l of protein kinase CK2 solution and 10. mu.l of substrate/ATP mixed solution were added sequentially to a white-bottomed 96-well plate to obtain a total volume of 25. mu.l of the reaction mixture, and the reaction was started by adding substrate/ATP to the kinase and incubated at room temperature for 60 min. Subsequently 25 μ l adp-Glo reagent was added to each reaction well to stop the reaction and consume the remaining ATP in each well, and incubated at room temperature for 40 min. And finally, adding 50 mul of kinase detection reagent into each hole, incubating for 30min at room temperature to convert the generated ADP into ATP, reacting with luciferase in the detection reagent to emit light, and detecting the light emission value by using a microplate reader. The inhibitory effect of different compounds on protein kinase CK2 at different concentrations was calculated by comparison with 100% inhibition of the reaction mixture without enzyme (negative control) and 0% inhibition with 5% DMSO (positive control).
The inhibition effect of the virtually screened candidate compound on the protein kinase CK2 is determined according to the experimental method, and the result is shown in figure 2, the candidate compounds I and II have good inhibition effect on the protein kinase CK2 in vitro, and the inhibition rates on the protein kinase CK2 are respectively 60% and 26% when the concentration of the candidate compounds is 64 mu M; the inhibition rate of the compound concentration of 256 mu M on the protein kinase CK2 is 72 percent and 40 percent respectively.
2) Anti-tumor proliferation assay for high kinase activity inhibitory compounds: the CCK-8 method is adopted for experimental determination. 2 x 10 to4Tumor cells in logarithmic growth phase were plated in 96-well plates at 5% CO2And incubating for 24h in an incubator at 37 ℃ until cells adhere to the wall, adding 100 mu l of inhibitors with different concentrations into each hole, incubating for 48h, adding 10 mu l of CCK-8 reagent, and measuring the absorbance of the mixture at 450nm by using an enzyme-labeling instrument after 2 h. Calculating the inhibition rate, making a nonlinear regression curve to obtain IC50The value is obtained.
As shown in FIG. 3, compounds I and II showed significant inhibitory activity, IC, against human lung cancer cells50The values are respectively: 23.08. mu.M and 8.81. mu.M.
In conclusion, the screening method of the protein kinase inhibitor provided by the invention can rapidly and efficiently screen out the lead compound of the protein kinase CK2 inhibitor from a large amount of compounds, and provides theoretical basis and experimental guidance for further structure optimization of the lead compound.
The above description is of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather as the invention is intended to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention.
Claims (9)
1. A method for screening a protein kinase inhibitor, comprising the steps of:
(1) obtaining a candidate compound by a virtual screening method;
(2) screening the candidate compound obtained in the step (1) for biological activity to obtain a target compound;
the virtual screening in the step (1) is a virtual screening method based on the combination of a pharmacophore model and molecular docking;
the biological activity screening in the step (2) is a kinase activity detection experiment and an anti-tumor cell proliferation experiment.
2. The method for screening a protein kinase inhibitor according to claim 1, wherein the pharmacophore model in step (1) is constructed by using Discovery Studio 4.0 software, and the molecular docking method is performed by using GOLD software.
3. The method for screening a protein kinase inhibitor according to claim 1, wherein the kinase activity assay in step (2) is an ADP-Glo kinase activity assay, and the anti-tumor cell proliferation assay is a CellCounting Kit-8(CCK-8) assay.
4. The method for screening a protein kinase inhibitor according to claim 1, which comprises the steps of:
s1.1, acquiring a three-dimensional structure of a Protein kinase CK2 and inhibitor eutectic crystal from a Protein data bank, and reserving and setting an active pocket according to a combined inhibitor;
s1.2, constructing a pharmacophore model based on receptor protein kinase CK2 by using the three-dimensional structure of the protein kinase CK2 and inhibitor eutectic obtained in the step S1.1, and performing primary screening based on a matching value on a small molecular compound library by using the pharmacophore model as an inquiry formula to obtain a primary screened compound library;
s1.3, according to the active pocket set in the step S1.1, performing molecular docking and grading sequencing on the compound screened in the step S1.2 by using molecular docking software, further screening to obtain a fine screening compound library, and evaluating an interaction mode of a docking molecule and CK2 to obtain a candidate compound;
s2, using the candidate compound obtained in the step S1.3 to carry out a kinase activity detection experiment and an anti-tumor cell proliferation experiment to obtain the target compound.
5. The method of claim 4, wherein in step S1.1, the three-dimensional structure of the co-crystal of protein kinase CK2 and the inhibitor is the three-dimensional structure of the co-crystal of protein kinase CK2 and the inhibitor in the existing protein database, or the three-dimensional structure of the co-crystal of protein kinase CK2 and other inhibitors;
in step S1.2, the construction method of the pharmacophore model comprises the following steps: constructing a pharmacophore model based on a receptor by using Discovery Studio 4.0, adopting determined molecular substances active and inactive to the receptor, performing model verification by using a Screen library module, wherein the obtained pharmacophore model contains 3-5 effective pharmacodynamic characteristic elements, and screening a small molecular compound library by using a matching degree value of more than 2.5-3.5 to obtain a primary screening compound library;
the small molecule compound library in the step S1.2 is a ChemBridge fragment library;
in step S1.3, the molecular docking procedure is preferably GOLD, or is replaced with other molecular docking software, such as: one or more of AutoDock vina, FlexX, or Glide;
when using GOLD for butt-joint scoring, using a Goldscore scoring function;
the fine screening compound library is preferably a compound which is scored to be 20-100, and the candidate compound is preferably 6-10 compounds which have similar action modes with the three-dimensional structure of the protein kinase CK2 and the inhibitor eutectic in the step S1.1.
6. The compound of interest obtained by the screening method of claims 1-5 is a compound having the structure of formula i, formula ii or a pharmaceutically acceptable salt thereof:
7. the use of a compound of formula i or formula ii as defined in claim 6, or a pharmaceutically acceptable salt thereof, for the preparation of an inhibitor of protein kinase CK2 or/and an anti-tumor medicament.
8. An application of a compound shown as a formula I and a formula II or pharmaceutically acceptable salt thereof in preparing a protein kinase CK2 inhibitor and/or an anti-tumor drug;
9. the use of the compound of claim 8 or a pharmaceutically acceptable salt thereof in the preparation of protein kinase CK2 inhibitors and/or antitumor drugs, wherein the compound of formula I and formula II has certain protein kinase CK2 inhibitory effects, and the inhibition rates of the compound at a compound concentration of 64 μ M on protein kinase CK2 are 60% and 26%, respectively; the inhibition rate of the compound concentration of 256 mu M on the protein kinase CK2 is 72 percent and 40 percent respectively;
the compounds shown in formula I and formula II have certain antitumor cell proliferation activity, and the tumor cell strain is human lung cancer A549 and IC thereof50The values are respectively: 23.08. mu.M and 8.81. mu.M.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112259175A (en) * | 2020-08-17 | 2021-01-22 | 杭州市第一人民医院 | Virtual screening method of IRAK1 kinase inhibitor and drug lead compound |
| CN112251820A (en) * | 2020-10-23 | 2021-01-22 | 绍兴文理学院 | VEGFR-2 inhibitor screening method, VEGFR-2 inhibitor and antitumor drug |
| CN112750496A (en) * | 2020-12-29 | 2021-05-04 | 大连理工大学 | Screening method of small molecule inhibitor of COVID-19 spinous process protein, active molecule screened by same and application |
| CN113380344A (en) * | 2021-06-11 | 2021-09-10 | 中国食品药品检定研究院 | FXR-based rapid screening method for hepatotoxic compounds in polygonum multiflorum |
| CN114224892A (en) * | 2021-11-23 | 2022-03-25 | 中国科学院深圳理工大学(筹) | TIPE2 inhibitor, application thereof, screening method and screening device |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170121318A1 (en) * | 2015-10-28 | 2017-05-04 | The Brigham And Women's Hospital, Inc. | Use of small molecule inhibitors to klf10 for modulation of t regulatory cells and cancer immunotherapy |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013022766A1 (en) * | 2011-08-05 | 2013-02-14 | Flynn Gary A | Preparation and methods of use for ortho-aryl 5- membered heteroaryl-carboxamide containing multi-targeted kinase inhibitors |
-
2019
- 2019-10-21 CN CN201910998324.1A patent/CN110718268B/en active Active
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170121318A1 (en) * | 2015-10-28 | 2017-05-04 | The Brigham And Women's Hospital, Inc. | Use of small molecule inhibitors to klf10 for modulation of t regulatory cells and cancer immunotherapy |
Non-Patent Citations (3)
| Title |
|---|
| HAOPENG SUN等: "Discovery and Design of Tricyclic Scaffolds as Protein Kinase CK2 (CK2) Inhibitors through a Combination of Shape-Based Virtual Screening and Structure-Based Molecular Modification.", 《J. CHEM. INF. MODEL.》, vol. 53, pages 2093 - 2102, XP055805696, DOI: 10.1021/ci400114f * |
| 无: "CAS：895954-84-4；CAS：831207-32-0", 《STN, REGISTRY》, pages 1 - 2 * |
| 陈文娟: "新型蛋白激酶 CK2 抑制剂的虚拟筛选及其抗癌活性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 05, pages 35 - 38 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112259175A (en) * | 2020-08-17 | 2021-01-22 | 杭州市第一人民医院 | Virtual screening method of IRAK1 kinase inhibitor and drug lead compound |
| CN112259175B (en) * | 2020-08-17 | 2023-05-12 | 杭州市第一人民医院 | Virtual screening method of IRAK1 kinase inhibitor |
| CN112251820A (en) * | 2020-10-23 | 2021-01-22 | 绍兴文理学院 | VEGFR-2 inhibitor screening method, VEGFR-2 inhibitor and antitumor drug |
| CN112251820B (en) * | 2020-10-23 | 2023-02-17 | 绍兴文理学院 | VEGFR-2 inhibitor screening method, VEGFR-2 inhibitor and antitumor drug |
| CN112750496A (en) * | 2020-12-29 | 2021-05-04 | 大连理工大学 | Screening method of small molecule inhibitor of COVID-19 spinous process protein, active molecule screened by same and application |
| CN113380344A (en) * | 2021-06-11 | 2021-09-10 | 中国食品药品检定研究院 | FXR-based rapid screening method for hepatotoxic compounds in polygonum multiflorum |
| CN113380344B (en) * | 2021-06-11 | 2023-09-19 | 中国食品药品检定研究院 | FXR-based rapid screening method for hepatotoxic compounds in Polygonum multiflorum |
| CN114224892A (en) * | 2021-11-23 | 2022-03-25 | 中国科学院深圳理工大学(筹) | TIPE2 inhibitor, application thereof, screening method and screening device |
Also Published As
| Publication number | Publication date |
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| WO2021077520A1 (en) | 2021-04-29 |
| CN110718268B (en) | 2023-11-03 |
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