阅读说明：本技术 代谢新化学实体肝cyp450亚型酶的筛选方法 (Method for screening liver CYP450 subtype enzyme for metabolizing new chemical entity ) 是由 李明 于 2020-12-09 设计创作，主要内容包括：本发明公开了一种代谢新化学实体肝CYP450亚型酶的筛选方法,其首先建立包含缓冲液、肝微粒体、第一底物、MgCl-2、抑制剂和NADPH的筛选体系,在筛选体系中进行新化学实体亚型酶的筛选反应,筛选反应分为加抑制剂组和不加抑制剂组,同时以第一底物进行验证试验,验证试验也分为加抑制剂组和不加抑制剂组,根据亚型酶筛选反应和验证试验的结果,对亚型酶筛选结果进行综合判断。本发明代谢新化学实体肝CYP450亚型酶的筛选方法能够有效减少假阴性与假阳性的发生率,保证筛选结果的可靠性。(The invention discloses a screening method of a liver CYP450 subtype enzyme for metabolizing a new chemical entity, which comprises the steps of firstly establishing a buffer solution, liver microsomes, a first substrate and MgCl 2 And the screening reaction of the new chemical entity subtype enzyme is carried out in the screening system, the screening reaction is divided into an inhibitor adding group and an inhibitor not adding group, meanwhile, a first substrate is used for carrying out a verification test, the verification test is also divided into the inhibitor adding group and the inhibitor not adding group, and the subtype enzyme screening result is comprehensively judged according to the subtype enzyme screening reaction and the result of the verification test. The screening method for metabolizing the new chemical entity liver CYP450 subtype enzyme can effectively reduce the incidence of false negative and false positive and ensure the reliability of the screening result.)

1. The screening method for metabolizing the new chemical entity liver CYP450 subtype enzyme is characterized by comprising the following steps:

step one, establishing a screening system:

in the medium comprising buffer, liver microsome, first substrate, MgCl₂Selecting an optimum reaction condition based on the metabolic rate of the first substrate in the reaction system of the inhibitor and NADPH, wherein the optimum reaction condition is the concentration of the inhibitor at which the inhibitor significantly inhibits the enzyme degrading the first substrate;

step two, subtype enzyme screening reaction and verification test:

under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂Mixing the inhibitors, adding NADPH to start a screening reaction, and detecting the content of a new chemical entity of the inhibitor group in a system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂Mixing inhibitor solvent, adding NADPH to start verification reaction, and detecting the content of new chemical entity without inhibitor in the system after the reaction is terminated;

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and the inhibitor, adding NADPH to start a verification reaction, and detecting the content of a first substrate of the inhibitor adding group in a system after the reaction is ended;

under the optimum reaction conditionBuffer solution, liver microsomes, first substrate, MgCl₂Mixing the inhibitor and a solvent, adding NADPH to start a verification reaction, and detecting the content of a first substrate without an inhibitor group in a system after the reaction is ended;

step three, judging the subtype enzyme screening result:

if the first substrate of the non-inhibitor group is metabolized, the first substrate of the inhibitor group is not metabolized, and the new chemical entity of the non-inhibitor group is metabolized, and the new chemical entity of the inhibitor group is not metabolized, indicating that the enzyme degrading the first substrate is involved in the metabolism of the new chemical entity.

2. The method for screening hepatic CYP450 subtype enzymes for metabolizing a new chemical entity according to claim 1, wherein said method for screening optimal reaction conditions in said first step comprises:

adopting an orthogonal test method, selecting a Tris buffer solution, taking the reaction condition when the content of the first substrate in the system is the lowest after the reaction is ended as the optimal reaction condition, and screening out the liver microsome, the first substrate and MgCl₂And the optimum concentration and optimum reaction time of NADPH;

in liver microsomes, first substrate, MgCl₂Under the optimum concentration of NADPH and the optimum reaction time, adopting Tris buffer solution and PBS buffer solution to carry out parallel test, and taking the buffer solution adopted in the group with the lowest content of the first substrate in the system after the reaction is terminated as the optimum buffer solution;

by adopting a single-factor test method, in liver microsome, first substrate, MgCl₂And the optimum concentration of NADPH, the optimum reaction time and the optimum buffer solution, and the optimum reaction concentration of the inhibitor is selected so that the concentration at which the inhibitor significantly inhibits the enzyme degrading the first substrate is the optimum concentration of the inhibitor.

3. The method of claim 2, wherein the optimal concentration of the inhibitor is selected to verify the specificity of the inhibitor, and the method comprises:

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and the inhibitor, adding NADPH to start reaction, and detecting the content of the first substrate in the system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsomes, a second substrate and MgCl₂Mixing the inhibitor and the inhibitor, adding NADPH to start reaction, and detecting the content of a second substrate in a system after the reaction is ended;

wherein the inhibitor corresponds to an enzyme that degrades a first substrate;

if neither the first substrate nor the second substrate is metabolized, indicating that the inhibitor is capable of inhibiting the enzyme that degrades the second substrate, then the concentration of the inhibitor should be reduced such that the inhibitor is capable of significantly inhibiting the enzyme that degrades the first substrate, but not the enzyme that degrades the second substrate.

4. The method of claim 2, wherein the step of screening hepatic CYP450 subtype enzyme for metabolizing a new chemical entity comprises screening hepatic microsomes, the first substrate, and MgCl under optimal conditions selected from the group consisting of Tris buffer, and the minimal amount of the first substrate in the system after the reaction has ended₂And the optimum concentration of NADPH and the optimum reaction time ", the first substrate series concentration is selected by: confirming K of an enzyme degrading a first substrate_mValue, K_mThe value, i.e.the first substrate concentration at which the enzymatic reaction rate is half the maximum reaction rate, was chosen as 1/3K_mValue sum 3K_mAs the two endpoints of the first substrate series of concentrations.

5. The method of claim 2, wherein the step of selecting the hepatic CYP450 subtype enzyme as the metabolic new chemical entity comprises performing a one-way assay on the hepatic microsome, the first substrate, and MgCl₂And optimum concentration of NADPH, optimum reaction time and optimum buffer solution, and screening the optimum reaction concentration of the inhibitor to obtain a concentration at which the inhibitor exhibits significant inhibition of the enzyme degrading the first substrateIn the "optimal concentration of the inhibitor", the series of concentrations of the inhibitor is selected by: k for confirming inhibitor_iValue, K_iThe concentration of free inhibitor corresponding to 50% of the enzyme bound by the inhibitor was chosen to be 2K_iValue sum 4K_iValues are given as the two endpoints of the inhibitor series concentration.

6. The method for screening hepatic CYP450 subtype enzyme for metabolizing new chemical entity according to claim 1, wherein the method for measuring the content of new chemical entity in the system in said second step is: after the reaction was terminated, it was measured by LC-MS/MS.

Technical Field

The invention relates to the technical field of in vitro rapid screening of liver CYP450 enzyme metabolic phenotype, in particular to a screening method of a liver CYP450 subtype enzyme which is a novel metabolic chemical entity.

Background

The research on the metabolism of the new chemical entity is an important content in the research and development process of the new chemical entity and has close relation with the speed and quality of the research and development of the new chemical entity. Therefore, the research on the drug metabolism of the new chemical entity has an indispensable important role in the research and development engineering of new drugs, and the research on the drug metabolism is important for understanding the change process of the drugs in vivo.

The in vitro metabolism method has short test period and controllable metabolism condition, and is convenient for evaluating the metabolism result. The liver is the main site of drug metabolism, and in vitro metabolism models are mostly based on the liver. Therefore, in vitro incubation of liver microsomes has become a popular in vitro metabolic study.

In the prior art, the subtype enzyme screening method by adopting liver microsomes has low accuracy, is easy to cause false positive or false negative, lacks verification tests and has low reliability of results.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a screening method for metabolizing a new chemical entity, namely a liver CYP450 subtype enzyme, which can reduce the incidence rate of false negative and false positive and ensure the reliability of a screening result.

The invention discloses a screening method of a liver CYP450 subtype enzyme for metabolizing a new chemical entity, which comprises the following steps:

step one, establishing a screening system:

in the medium comprising buffer, liver microsome, first substrate, MgCl₂Selecting an optimum reaction condition based on the metabolic rate of the first substrate in the reaction system of the inhibitor and NADPH, wherein the optimum reaction condition is the concentration of the inhibitor at which the inhibitor significantly inhibits the enzyme degrading the first substrate;

step two, subtype enzyme screening reaction and verification test:

under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂Mixing the inhibitors, adding NADPH to start a screening reaction, and detecting the content of a new chemical entity of the inhibitor group in a system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂Mixing inhibitor solvent, adding NADPH to start verification reaction, and detecting the content of new chemical entity without inhibitor in the system after the reaction is terminated;

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and the inhibitor, adding NADPH to start a verification reaction, and detecting the content of a first substrate of the inhibitor adding group in a system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and a solvent, adding NADPH to start a verification reaction, and detecting the content of a first substrate without an inhibitor group in a system after the reaction is ended;

step three, judging the subtype enzyme screening result:

if the first substrate of the non-inhibitor group is metabolized, the first substrate of the inhibitor group is not metabolized, and the new chemical entity of the non-inhibitor group is metabolized, and the new chemical entity of the inhibitor group is not metabolized, indicating that the enzyme degrading the first substrate is involved in the metabolism of the new chemical entity.

Preferably, the method for screening the optimal reaction conditions in the first step comprises:

adopting an orthogonal test method, selecting a Tris buffer solution, taking the reaction condition when the content of the first substrate in the system is the lowest after the reaction is ended as the optimal reaction condition, and screening out the liver microsome, the first substrate and MgCl₂And the optimum concentration and optimum reaction time of NADPH;

in liver microsomes, first substrate, MgCl₂Under the optimum concentration of NADPH and the optimum reaction time, adopting Tris buffer solution and PBS buffer solution to carry out parallel test, and taking the buffer solution adopted in the group with the lowest content of the first substrate in the system after the reaction is terminated as the optimum buffer solution;

by adopting a single-factor test method, in liver microsome, first substrate, MgCl₂And the optimum concentration of NADPH, the optimum reaction time and the optimum buffer solution, and the optimum reaction concentration of the inhibitor is selected so that the concentration at which the inhibitor significantly inhibits the enzyme degrading the first substrate is the optimum concentration of the inhibitor.

Further preferably, in the screening of the optimal concentration of the inhibitor, the specificity of the inhibitor needs to be verified, and the method for verifying the specificity of the inhibitor comprises the following steps:

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and the inhibitor, adding NADPH to start reaction, and detecting the content of the first substrate in the system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsomes, a second substrate and MgCl₂Mixing the inhibitor and the inhibitor, adding NADPH to start reaction, and detecting the content of a second substrate in a system after the reaction is ended;

wherein the inhibitor corresponds to an enzyme that degrades a first substrate;

if neither the first substrate nor the second substrate is metabolized, indicating that the inhibitor is capable of inhibiting the enzyme that degrades the second substrate, then the concentration of the inhibitor should be reduced such that the inhibitor is capable of significantly inhibiting the enzyme that degrades the first substrate, but not the enzyme that degrades the second substrate.

Further preferably, the step "adopts an orthogonal test method, selects Tris buffer solution, takes the reaction condition when the content of the first substrate in the system is the lowest after the reaction is finished as the optimal reaction condition, and screens out the liver microsome, the first substrate and MgCl₂And the optimum concentration of NADPH and the optimum reaction time ", the first substrate series concentration is selected by: confirming K of an enzyme degrading a first substrate_mValue, K_mThe value, i.e.the first substrate concentration at which the enzymatic reaction rate is half the maximum reaction rate, was chosen as 1/3K_mValue sum 3K_mAs the two endpoints of the first substrate series of concentrations.

Further preferably, the step "uses a one-factor assay in liver microsomes, first substrate, MgCl₂And the optimum concentration of NADPH, the optimum reaction time and the optimum buffer solution, and the optimum reaction concentration of the inhibitor is selected so that the concentration at which the inhibitor significantly inhibits the enzyme degrading the first substrate is the optimum concentration of the inhibitor, and the concentration of the inhibitor is selected by: k for confirming inhibitor_iValue, K_iThe concentration of free inhibitor corresponding to 50% of the enzyme bound by the inhibitor was chosen to be 2K_iValue sum 4K_iValues are given as the two endpoints of the inhibitor series concentration.

Preferably, the method for detecting the content of the new chemical entity in the system in the second step comprises the following steps: after the reaction was terminated, it was measured by LC-MS/MS.

The invention has the following beneficial effects:

the invention verifies and optimizes the liver microsome, the concentration of the first substrate, the concentration of the inhibitor and the reaction time in the screening system, effectively reduces the incidence rate of false negative and false positive, improves the reliability of the identification result of the metabolic liver CYP450 subtype enzyme of the new chemical entity, and provides a reliable basis for promoting the research and development of new drugs.

When the new chemical entity metabolic liver CYP450 subtype enzyme is screened, a screening test and a plurality of verification tests are simultaneously arranged, so that the reliability of the screening result is ensured, and when a negative result appears, the reason of the negative result can be quickly judged.

In order to make the aforementioned and other objects, features and advantages of the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment discloses a screening method for metabolizing a new chemical entity liver CYP450 subtype enzyme, which comprises the steps of firstly establishing a screening system, carrying out subtype enzyme screening reaction and verification test in the screening system, and judging a subtype enzyme screening result according to the results of the subtype enzyme screening reaction and the verification test.

In the assay, both the new chemical entity and the first substrate prototype component were tested using LC-MS/MS. Before LC-MS/MS detection is used, LC-MS/MS needs to be optimized and methodological verification is carried out, and contents recorded in pharmacopoeia 9012 are specifically referred.

The screening method of the liver CYP450 subtype enzyme for metabolizing the new chemical entity comprises the following specific steps:

step one, establishing a screening system:

this example sets up a screening system that enables screening of the major subtype of hepatic CYP450 enzymes. In the establishment of the screening system, specific substrates and corresponding inhibitors corresponding to the main subtypes of the liver CYP450 enzyme are selected, and a screening system is established for each substrate. That is, the optimum screening conditions for the hepatic CYP450 subtype enzyme corresponding to the selected substrate are screened.

The method for establishing each substrate screening system is the same, and the first substrate is taken as an example and is explained in detail below:

1. adopting an orthogonal test method, selecting a Tris buffer solution, taking the reaction condition when the content of the first substrate in the system is the lowest after the reaction is ended as the optimal reaction condition, and screening out the liver microsome, the first substrate and MgCl₂And the optimum concentration of NADPH and the optimum reaction time. Wherein NADPH is used to initiate the reaction, in the liver microsomes, the first substrate and MgCl₂After dissolving in Tris buffer, NADPH was added. The first substrate is: a substrate specific for the enzyme to which the inhibitor corresponds.

The serial concentrations of liver microparticles were set at 0.2mg/mL, 0.5mg/mL, and 1.0 mg/mL.

The first substrate series of concentrations was selected by: confirming K of an enzyme degrading a first substrate_mValue, K_mThe value is the first substrate concentration corresponding to half the maximum reaction rate of the enzymatic reaction. The substrates used in the tests are all those known in the art, the K_mThe value is also known, K_mValues can be obtained by simply looking up published literature. But due to K_mThe values are experimental data, and the values can be different in different documents, and the searched K is taken_mOne median in the value as K adopted in this example_mValue, reselect 1/3K_mValue sum 3K_mAs the two endpoints of the first substrate series of concentrations.

NADPH and MgCl₂The concentration series of (2) is set to be 10mmol/L, 20mmol/L and 40 mmol/L. NADPH and MgCl₂The optimal concentration is selected according to the following: the minimum concentration is chosen, provided that the reaction is ensured to occur to a maximum extent.

The reaction time was 5min, 10min, 20min, 30min, 45min, and 60 min. The reaction time is selected according to the following: the prototype composition of the first substrate was reduced by 80% or more, and the time point at which the reduction by 80% was achieved was defined as the optimum reaction time.

2. In liver microsomes, first substrate, MgCl₂And optimum concentration and optimum reaction of NADPHAnd (3) performing parallel tests by using Tris buffer solution and PBS buffer solution, and comparing the content of the first substrate in the system in the Tris buffer solution group and the PBS buffer solution group after the reaction is terminated, wherein the buffer solution used in the lowest group is the optimal buffer solution.

3. By adopting a single-factor test method, in liver microsome, first substrate, MgCl₂And the optimum concentration of NADPH, the optimum reaction time, and the optimum buffer solution, and screening the optimum reaction concentration of the inhibitor. The inhibitor corresponds to an enzyme that degrades the first substrate.

The optimal response concentration of the inhibitor is screened according to the following steps: the optimum concentration of the inhibitor is the concentration at which the inhibitor exhibits significant inhibition of the enzyme degrading the first substrate. I.e., after the reaction is terminated, the amount of the first substrate of the inhibitor-added group is significantly higher than the amount of the first substrate of the inhibitor-not-added group, i.e., indicating that the enzyme degrading the first substrate is significantly inhibited by the inhibitor.

The selection method of the inhibitor series concentration comprises the following steps: k for confirming inhibitor_iThe value is obtained. K_iThe value of 50% corresponds to the concentration of free inhibitor when the enzyme is bound by the inhibitor. The inhibitors used in the tests are those defined in the prior art, the K of which_iThe values are also well known. K_iValues can be obtained by simply looking up published literature. But due to K_iTaking the value as test data, wherein the value can be different in different documents, and taking the looked-up K_iOne median in the value as K adopted in this example_iValue, select 2K_iValue sum 4K_iValues are given as the two endpoints of the inhibitor series concentration.

In screening the optimal concentration of the inhibitor, the specificity of the inhibitor needs to be verified, and the verification method comprises the following steps:

under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂Mixing the inhibitor and the inhibitor, adding NADPH to start reaction, and detecting the content of the first substrate in the system after the reaction is ended;

under the optimal reaction conditions, buffer solution, liver microsomes, a second substrate and MgCl₂Mixing the inhibitor, adding NADPH to start reaction, and stopping reactionThen, detecting the content of the second substrate in the system;

comparing the amount of the first substrate in the system before the reaction and after the reaction is terminated, wherein if the amount of the first substrate in the system is not reduced, it indicates that the first substrate is not metabolized, and if the amount of the first substrate is significantly reduced, it indicates that the first substrate is metabolized.

And comparing the content of the second substrate in the system before the reaction and after the reaction is terminated, wherein if the content of the second substrate in the system is not reduced, the second substrate is not metabolized, and if the content of the second substrate is obviously reduced, the second substrate is metabolized.

The first substrate is metabolized while the second substrate is not metabolized, indicating that the inhibitor inhibits only the subtype enzyme that degrades the first substrate.

If neither the first substrate nor the second substrate is metabolized, indicating that the inhibitor is capable of inhibiting the subtype enzyme that degrades the second substrate, then the concentration of the inhibitor should be reduced such that the inhibitor is capable of significantly inhibiting the subtype enzyme that degrades the first substrate, but not inhibiting the subtype enzyme that degrades the second substrate.

In other words, the screening of the optimal concentration of the inhibitor mainly comprises the steps of screening a preliminary optimal concentration by a single-factor test method, performing inhibitor specificity verification according to the preliminary optimal concentration, and adjusting the preliminary optimal concentration according to the inhibitor specificity verification result to obtain a final optimal concentration, wherein the final optimal concentration is the optimal concentration of the inhibitor adopted in the embodiment.

4. Obtaining the optimal reaction conditions of the screening system through the screening and adjusting processes, wherein the optimal reaction conditions comprise the optimal reaction time, the optimal buffer solution type, the optimal concentration of the liver microsomes, the optimal concentration of the first substrate, and MgCl₂Optimal concentration, inhibitor optimal concentration and NADPH optimal concentration.

NADPH in the screening system is used to start the reaction and needs to be added after the other components are well mixed. When the reaction was terminated, acetonitrile as a terminator was added to terminate the reaction.

In the screening system of the embodiment, if the concentration of the liver microsomes is too high, the metabolic capability is too strong, so that a false positive result is caused; if the concentration of liver microsomes is too low, the metabolic capacity is too weak, and a false negative result is caused. If the concentration of the first substrate is too high, the metabolism of the liver microsome to the first substrate is not obvious, and a false negative result appears; if the concentration of the first substrate is too low, the inhibitory effect of the inhibitor on the enzyme is not obvious, and a false positive result appears. If the concentration of the inhibitor is too high, the inhibition capability is too strong, and a false positive result appears; if the concentration of the inhibitor is too low, the inhibition effect is not obvious, and a false negative result appears. NADPH is used for supplying energy to a system, so that the reaction requirement can be met, and if the concentration is too high, waste is caused. If the reaction time is too long, a false positive result may occur, and if the reaction time is too short, a false negative result may occur. This example screens for optimal reaction conditions, avoiding the appearance of false positive and false negative results.

Positive means that the subtype enzyme is involved in the metabolism of a new chemical entity. Negative means that the subtype enzyme is not involved in the metabolism of a new chemical entity.

Step two, subtype enzyme screening reaction and verification test:

the hepatic CYP450 subtype enzymes involved in NCE metabolism generally include a plurality. In the subtype enzyme screening reaction and the verification test, the screening system in the step one is adopted to screen main subtype enzymes in sequence to obtain the liver CYP450 subtype enzyme participating in NCE metabolism. The screening reaction and the validation test procedure for each of the hepatic CYP450 subtype enzymes are the same, and are detailed below with reference to the hepatic CYP450 subtype enzyme degrading the first substrate:

under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂And mixing the inhibitors, adding NADPH to start a screening reaction, adding a terminator to terminate the reaction after the reaction time is reached, and determining the content of the new chemical entity in the system by adopting LC-MS/MS to obtain the metabolic result of the new chemical entity added with the inhibitors.

Under the optimal reaction conditions, buffer solution, liver microsomes, new chemical entities and MgCl are added₂And mixing the inhibitor and the solvent, adding NADPH to start a verification reaction, and adding a terminator to stop the reaction after the reaction time is up. Determining the content of the new chemical entity in the system by LC-MS/MS to obtain the group without inhibitorMetabolic outcome of the new chemical entity of (1). Wherein, the inhibitor is dissolved in the inhibitor solvent, and when the inhibitor is not added, the same inhibitor solvent needs to be added to ensure the accuracy of the reaction result.

Under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂And mixing the inhibitor, adding NADPH to start a verification reaction, and adding a terminator to stop the reaction after the reaction time is reached. And detecting the content of the first substrate of the inhibitor adding group in the system by adopting LC-MS/MS to obtain the metabolism result of the first substrate of the inhibitor adding group.

Under the optimal reaction conditions, buffer solution, liver microsome, first substrate and MgCl are added₂And mixing the inhibitor and the solvent, adding NADPH to start a verification reaction, and adding a terminator to stop the reaction after the reaction time is up. And detecting the content of the first substrate of the inhibitor-free group in the system by adopting LC-MS/MS (liquid chromatography-mass spectrometry) to obtain the metabolism result of the first substrate of the inhibitor-free group.

When the screening test is carried out, factors such as enzyme activity, reagent errors, manual operation and the like easily influence the screening result, and the false negative and false positive detection results are avoided by setting the verification test, so that the screening result is accurate and reliable.

The optimal buffer solution is one of Tris buffer solution or PBS buffer solution, and the optimal buffer solution obtained by screening in the step one is selected.

Step three, judging the subtype enzyme screening result:

the substrates used in this example are well established in the art and their metabolic results in the screening system are also predictable. Therefore, if the metabolic result of the used substrate in the screening system reaches the expected value, the screening is considered to have no problem, the test is successful, and the screening reaction result of the new chemical entity is accepted.

After the reaction is terminated in the second step, the contents of the first substrates of the inhibitor-free group and the inhibitor-added group in the system are compared, and if the content of the first substrates of the inhibitor-free group is lower than that of the inhibitor-added group by half, the first substrates of the inhibitor-free group are metabolized, and the first substrates of the inhibitor-added group are not metabolized.

And after the reaction in the second step is ended, comparing the content of the new chemical entities in the inhibitor-free group and the inhibitor-added group in the system, wherein if the content of the new chemical entities in the inhibitor-free group is lower than that in the inhibitor-added group by half, the new chemical entities in the inhibitor-free group are metabolized, and the new chemical entities in the inhibitor-added group are not metabolized.

If the first substrate of the inhibitor-added group is metabolized, the screening reaction results are not accepted regardless of whether the new chemical entity of the inhibitor-added group is metabolized.

If the first substrate of the inhibitor-free group is not metabolized, the screening reaction results are not accepted regardless of whether the new chemical entity of the inhibitor-free group is metabolized.

The reasons for the non-acceptance of the results of the screening reaction are: the first substrate and the inhibitor are associated with the same enzyme, and the inhibitor must inhibit the enzyme that degrades the first substrate, so that the first substrate of the inhibitor-added group must not be metabolized, and the same first substrate of the inhibitor-free group must be metabolized.

If the first substrate of the inhibitor-free group is metabolized and the first substrate of the inhibitor-added group is not metabolized, the metabolic results of the new chemical entity are considered as follows:

if the new chemical entity without the inhibitor group is metabolized, the new chemical entity with the inhibitor group is also metabolized, indicating that the enzyme that degrades the first substrate does not contribute to the metabolism of the new chemical entity, i.e., indicating that the enzyme that degrades the first substrate is not involved in the metabolism of the new chemical entity, which is not the metabolic liver CYP450 subtype enzyme to be screened in this example.

If the new chemical entity without the inhibitor group is metabolized and the new chemical entity with the inhibitor group is not metabolized, it indicates that the enzyme degrading the first substrate is involved in the metabolism of the new chemical entity, which is the metabolic hepatic CYP450 subtype enzyme to be screened in this example.

If neither the new chemical entity of the no inhibitor group nor the new chemical entity of the inhibitor group is metabolized, it is an indication that the new chemical entity is not metabolized by the hepatic CYP450 subtype enzyme.

If the new chemical entity of the inhibitor-free group is not metabolized and the new chemical entity of the inhibitor-added group is metabolized, the test result is considered against the conventional theory, and the test is repeated without accepting the test result.

The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.