阅读说明：本技术 一种壳聚糖水解酶可逆抑制保护剂的筛选方法 (Screening method of chitosan hydrolase reversible inhibition protective agent ) 是由 许向阳 宋在伟 殷允超 种法国 宋均营 黄家超 叶王立 王鹏 于 2020-12-25 设计创作，主要内容包括：本发明要解决的技术问题是提供一种壳聚糖水解酶的可逆抑制保护剂的筛选方法,弥补现有技术存在的不足,适用于壳聚糖水解酶在工业酶制剂上的应用,提高其稳定性。本发明的设计思路如下：首先建立已知的可逆抑制保护剂训练集,根据已有的壳聚糖水解酶结构数据,进行分子对接并排序,并结合测试结果进行验证,确定方法的可靠性。后建立筛选规则和筛选模型,使用该规则和模型分别对小分子化合物数据库进行筛选、排序和打分,逐渐缩小范围,最终根据由上到下几次排序的结果,确定可以作为壳聚糖水解酶可逆抑制保护剂的化合物。(The invention aims to solve the technical problem of providing a screening method of a reversible inhibition protective agent of chitosan hydrolase, which makes up the defects of the prior art, is suitable for application of the chitosan hydrolase in industrial enzyme preparations and improves the stability of the chitosan hydrolase. The design idea of the invention is as follows: firstly, establishing a known reversible inhibition protective agent training set, carrying out molecular docking and sequencing according to the existing structural data of the chitosan hydrolase, and verifying by combining a test result to determine the reliability of the method. And then establishing a screening rule and a screening model, respectively screening, sorting and scoring the small molecule compound database by using the rule and the model, gradually reducing the range, and finally determining the compound which can be used as the chitosan hydrolase reversible inhibition protective agent according to the results of sorting from top to bottom.)

1. A screening model for a reversible inhibition protective agent of chitosan hydrolase is characterized in that the screening model is established by the following method:

1) determining the category of the reversible inhibition protective agent and the inhibition constant of the chitosan hydrolase according to the structural data of the chitosan hydrolase, and determining the compounds of the training set;

2) performing molecular docking on the compounds in the training set in the step 1) and chitosan hydrolase, and correcting an inhibition constant of the activity of the chitosan hydrolase by using the reversible inhibition protective agent in the step 1);

3) and (3) making a screening rule according to the binding free energy and the reversible inhibition constant of the compound in the training set and the chitosan hydrolase obtained in the step 2) and the screening result of the compound in the training set in the step 1), and establishing a screening model.

2. The screening model of claim 1, wherein the compounds of step 1) entering the training set comprise 2, 4-dinitroanisole (2, 4-dinitro-1-fluorobenzene), 2-Hydroxy-5-nitrobenzyl bromide (2-Hydroxy-5-nitrobenzyl bromide), Sodium acetate (Sodium acetate), benzalkonium chloride (benzalkonium chloride), ethyl acetiminate (ethyl acetimidate), disodium Edetate (EDTA), glucosamine (glucosamine), guanidine hydrochloride (guanidium hydrochloride), cysteine (cysteine), and iodoacetate (monoiodoacetate).

3. The screening model of claim 1, wherein the molecular docking method of step 2) is performed by using REDUCE, Autodock Tools and Autodoc 4.

4. The screening model of claim 3, wherein the molecular docking method of step 2) comprises adding hydrogen bonds and Gaussian charges to the chitosan hydrolase and the inhibitor, and docking the molecule with the chitosan hydrolase active site region with complete flexibility.

5. The screening model of claim 1, wherein the rules for screening in step 3) are as follows: ei is more than or equal to-6.000 and less than or equal to-5.000, and Ki is more than or equal to 50 and less than or equal to 200.

6. The screening model of claim 1, wherein said screening model is a potent pharmacophore model having 2 hydrophobic centers, and 3 hydrogen bond acceptors and 5 donors.

7. A method of screening for a protective agent against reversible inhibition of chitosan hydrolase using the screening model of any one of claims 1 to 6.

8. The screening method according to claim 7, wherein the compounds to be screened are composed into a compound pool A according to the screening rules established by the screening model according to any one of claims 1 to 6, and subjected to rank ordering, the compounds in the compound pool A are reordered according to theoretical binding free energy and theoretical reversible inhibition constant, the molecules ranked at the top 2000 position are jointly scored using an X-score scoring function, the affinity with the target protein is calculated, and the compounds with the score value of more than 5.0 are taken to be composed into a compound pool B; and after the compound pool B is obtained, performing fitting sequencing by using a screening model, and screening the reversible inhibition protective agent of the chitosan hydrolase.

Technical Field

The invention belongs to the technical field of active substance screening, and particularly relates to a screening method of a chitosan hydrolase reversible inhibition protective agent.

Background

The enzyme has the characteristics of small dosage, high catalytic efficiency and strong specificity. After the rapid development of the enzyme preparation industry for more than half a century, the enzyme preparation industry has become a high-technology industry with high vitality and is widely applied to industries such as food, textile, washing, paper making, feed, biological energy and the like. At present, compared with the production and manufacturing process of similar products, the liquid enzyme preparation technology saves water by about 30-50 percent and saves energy by more than 50 percent, and is more in line with the development requirement of low-carbon economy; it is needless to say that a high-activity, high-purity, liquid enzyme will be the development direction of enzyme preparations in the future.

The active function of the enzyme is determined by the complete and strict conformation of the molecular structure, and the change of environmental factors is easy to cause the spatial structure of the enzyme to be damaged, thereby losing the original biological activity. Some small molecular substances can interact with amino acid residues in the active center of the enzyme and lock the amino acid residues, and space limitation is carried out on the conformation of the enzyme, so that the enzyme enters a dormant state. Then under certain activation condition, the acting force of the small molecular substance and the enzyme active center is weakened, so that the small molecular substance is released, the functional group of the active center regains free extension, and the activity of the enzyme is recovered, which is the basic principle of the action of the enzyme reversible inhibitor. In the early 60 s of the 20 th century, Umezawa originally proposed the concept of enzyme inhibition, developed to date, and mainly used in the field of drug screening.

The industrial enzyme reversible inhibitor has no ready-made specific screening model and referential structure information database, is a difficult problem in the research field all the time, and has not been broken through at home. However, no effective method exists in the technical field of screening of the chitosanase reversible inhibition protective agent.

Disclosure of Invention

The invention aims to provide a screening method of a reversible inhibition protective agent of chitosan hydrolase, wherein the screened reversible inhibition protective agent can improve the stability of the chitosan hydrolase; thereby making up the defects of the prior art.

The method comprises the steps of firstly establishing a known reversible inhibition protective agent training set, carrying out molecular docking and sequencing according to the existing structural data of the chitosan hydrolase, and verifying by combining a test result to determine the reliability of the method. And then establishing a screening rule and a screening model, respectively screening, sorting and scoring the small molecule compound database by using the rule and the model, gradually reducing the range, finally determining a compound which can be used as a chitosan hydrolase reversible inhibition protective agent according to the results of sorting from top to bottom, and carrying out further experimental verification.

The screening method of the reversible inhibition protective agent of chitosan hydrolase provided by the invention comprises the following steps:

1) determining the category of the reversible inhibition protective agent and the inhibition constant of the chitosan hydrolase according to the structural data of the chitosan hydrolase, and determining the compounds of the training set;

2) performing molecular docking on the compounds in the training set in the step 1) and chitosan hydrolase, and correcting an inhibition constant of the activity of the chitosan hydrolase by using the reversible inhibition protective agent in the step 1);

3) formulating a screening rule and establishing a screening model according to the binding free energy and the reversible inhibition constant of the compound in the training set and the chitosan hydrolase obtained in the step 2) and the screening result of the compound in the training set in the step 1);

4) screening according to the screening rule formulated in the step 3) and the established screening model.

Further, the compound entering the training set in step 1) is 2, 4-dinitroanisole (2, 4-dinitro-1-fluorobenzene), 2-Hydroxy-5-nitrobenzyl bromide (2-Hydroxy-5-nitrobenzyl bromide), Sodium acetate (Sodium acetate), benzalkonium chloride (benzalkonium chloride), ethyl acetimidate (ethyl acetate), disodium Ethylenediaminetetraacetate (EDTA), glucosamine (gluconamide), guanidine hydrochloride (guanidinium hydrochloride), cysteine (cysteine), iodoacetic acid (monofluoroacetate); these compounds are inhibitors of chitosanhydrolase, including both reversible and irreversible inhibitors, which bind to proteins to varying degrees

Further, the molecular docking method in the step 2) comprises the following steps: the method is jointly completed by REDUCE, Autodock Tools and Autodoc4, firstly hydrogen bonds and Gaussian charges are added to chitosan hydrolase and an inhibitor, and then a completely flexible docking method of molecules and the active site region of chitosan hydrolase is adopted;

furthermore, the rule of screening is that-6.000 is less than or equal to Ei is less than or equal to-5.000, and 50 is less than or equal to Ki is less than or equal to 200, and the rule is used as the screening rule.

Further, the established screening model is an effective pharmacophore model, and the model is provided with 2 hydrophobic centers, 3 hydrogen bond acceptors and 5 donors; excluded on an Xscore scale, under 5.0 points;

further, the screening method comprises the steps of forming a compound pool A from the compounds screened from the small molecule database according to the screening rule formulated in the step 3), and carrying out sequencing and scoring,

reordering the compounds in the compound pool A according to theoretical combination free energy and theoretical reversible inhibition constant, taking the molecules ranked at the first 2000 position, using an X-score scoring function to score jointly, calculating the affinity with the target protein, and taking the compounds with the scoring value larger than 5.0 to form a compound pool B;

after compound pool B was obtained, the screening model was used for fitting ranking.

The screening method of the reversible inhibition protective agent of chitosan hydrolase, which is established by the invention, has the following advantages: (1) the screening method is rapid, the number of candidate molecules can be rapidly reduced, and the waste of time and test materials is reduced; (2) the method is reliable, because the establishment of the screening rule and the model is carried out on the basis of the existing inhibitor training set, the final screening result is also verified by using experimental data, the theory and the experimental result are mutually complemented, and the reliability is high; (3) the targeting is good, and the molecular docking used in the method is based on modeling of existing structural data of chitosan hydrolase (Jun Sheng, Xiaoofeng Ji, Yuan Zheng, etc. Improvement in the mobility of bacteria by the construction of specific diagnostic blocks [ J ]. Biotechnology Letters,2016.) and has strong targeting. The screening method is suitable for application of chitosan hydrolase in industrial liquid enzyme preparations.

Drawings

FIG. 1: a structure model diagram of the chitosan hydrolase;

FIG. 2: and (3) building a structural model diagram after the chitosan hydrolase is combined with the inhibition protective agent.

Detailed Description

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.

The method for calculating the inhibition constant of the chitosan hydrolase comprises the following steps:

chitosan hydrolase catalyzed Michaelis constant (K)_m) Using a Lineweaver-Burk double reciprocal mapping method to achieve the inhibition constant K_iValues were obtained by Dixon mapping. The Lineweaver-Burk equation can be written as:

obtaining after secondary derivation:

from which the suppression constant K can be determined_i

Example 1 screening method for protective agents against reversible inhibition of chitosanase

A method for screening a protective agent for the reversible inhibition of chitosanolytic enzyme comprising the steps of:

(1) according to the sequence structure of known chitosanhydrolase, structural data of the inhibitor, and experimental data of the reaction of the enzyme with small molecule compounds (table 1), the inhibitor comprises Sodium acetate (Sodium acetate), benzalkonium chloride (benzalkonium chloride), ethyl acetimidate (ethyl acetate), disodium Edetate (EDTA), glucosamine (glucoamine), guanidine hydrochloride (guanidium hydrochloride), cysteine (cysteine), iodoacetic acid (monooxyacetate);

table 1: table of inhibition constants for reaction of chitin hydrolase inhibitors

The inhibition constant (Ki) reflects the degree of binding of the compound to the enzyme, and in the 18 compounds, the binding to chitosanhydrolase is too strong (Ki <0.3), which easily results in complete denaturation of the enzyme molecule, and the remainder enters the training set of reversible inhibitory protectants.

(2) Molecular docking of compounds in the training set with chitosanhydrolase

The molecular docking method used was: this is done using REDUCE, Autodock Tools and Autodoc 4. The hydrogen bonds were added first by the REDUCE program, followed by the addition of the Gaussian charge using the Autodock Tools script. Selecting a coordinate point (12.403, -20.749, 24.022) as a center,the space area in the range of (1) is a butted grid point area, and grid points are defined by using an AutoGrid. Docking was performed using Autodock4 using a docking method where the inhibitor and the active site region of the enzyme were completely flexible. The scoring adopts a method of combining self scoring and Xscore scoring of Autodock 4.

The results after docking are shown in table 2.

Table 2: training set theory after molecular docking combines free energy (E) and theory reversible inhibition constant (Ki)

(3) Establishing and perfecting a screening rule and a screening model;

after obtaining the theoretical binding free energy (Ei, kcal/mol) and reversible inhibition constant (Ki, μ M) of the compounds in the training set with chitosan hydrolase, referring to the data of the experimental results in Table 1, the values of the docking energy and the theoretical reversible inhibition constant (Ki) of these compounds need to satisfy the selection conditions (-6.000. ltoreq. Ei.ltoreq. -5.000, 50. ltoreq. Ki.ltoreq.200), so this is used as a screening rule, within which the reversible inhibition result is positive.

The screening model established in this way is an effective pharmacophore model, which has 2 hydrophobic centers, 3 hydrogen bond acceptors and 5 donors; excluded on an Xscore scale, under 5.0 points;

(4) screening the mixture to obtain a high-quality product,

firstly, using the obtained screening rule (-6.000-5.000 Ei, 50-200 Ki) to primarily screen about 1900 ten thousand small molecule compounds in a ZINC database, obtaining 13000 compounds, and forming a compound pool A.

Reordering the compounds in pool A according to theoretical binding free energy (Ei,) and theoretical reversible inhibition constant (Ki), taking the molecules ranked at the first 2000 position and jointly scoring by using X-score scoring function, calculating the affinity with the target protein, taking the score value larger than 5.0, and composing the total 283 compounds into pool B.

And after the compound pool B is obtained, fitting of a screening model is carried out. Using the screening model obtained above to match 283 compounds, with the elimination of clusters that did not match the model or that were more than 2-fold more than the number of model molecular clusters, the most structurally matched compound ranked in the top 3 positions was finally obtained (table 3):

table 3: ten compounds matched with screening model

These compounds may be used as compound molecules as reversible inhibition protectors for chitosan hydrolases.

Example 2 docking simulation of the Compound ZINC01006243 with Chitosan hydrolase

A first-scoring compound 5,7-Diphenyl-1, 7-dihydro-pyrolo [2,3-d ] pyrimidine-2,4-dithione is selected for research, is dissolved in ethanol to prepare a 1mM solution, and is added into a concentrated chitosan hydrolase solution (8000u/ml) according to the amount of 25 mu M, so that the enzyme activity is inhibited, and the activity is kept by 18.76%. After 30 days at 40 ℃ the mixture was stored according to a 1: after 10 times of dilution, the detection result shows that the activity is 652u/ml, the activity is recovered, and the relative enzyme activity exceeds 80%.

The results show that the theory and the experimental results of the screening method established by the invention are complementary, and the reliability is high.