阅读说明：本技术 化合物的靶点的鉴定方法、化合物与靶标相互作用的检测方法和化合物药效的评价方法 (Method for identifying target of compound, method for detecting interaction between compound and target, and method for evaluating drug effect of compound ) 是由 戴伦治 王秀轩 王新媛 于 2019-10-12 设计创作，主要内容包括：本发明公开了化合物的靶点的鉴定方法、化合物与靶标相互作用的检测方法和化合物药效的评价方法,涉及药物靶点鉴定技术领域。具体而言,将经过目标化合物处理后细胞或者动物的蛋白或蛋白酶解产物与捕获剂孵育；所述捕获剂为可特异性识别并结合所述目标化合物和/或含有目标化合物的结合物的抗体。该鉴定方法采用捕获剂特异性地获取或富集蛋白或多肽溶液中的目标化合物和/或含有目标化合物的结合物,通过质谱分析,鉴定捕获剂捕获目标化合物的结合物是否是/或属于靶标蛋白、捕获的靶标蛋白是什么,以及捕获的靶标蛋白的数量和丰度,该鉴定方法无需对目标化合物结构进行修饰,直接有效特异性地富集并鉴定出目标化合物的靶标。(The invention discloses a compound target identification method, a compound target interaction detection method and a compound efficacy evaluation method, and relates to the technical field of drug target identification. Specifically, the protein or proteolysis product of the cell or animal treated by the target compound is incubated with a capture agent; the capture agent is an antibody that specifically recognizes and binds the target compound and/or a conjugate containing the target compound. The identification method adopts a capture agent to specifically obtain or enrich a target compound and/or a conjugate containing the target compound in a protein or polypeptide solution, and identifies whether the conjugate of the target compound captured by the capture agent belongs to the target protein or not, what the captured target protein belongs to and the quantity and abundance of the captured target protein through mass spectrometry.)

1. A method for identifying a target of a compound, comprising: incubating the protein of the cell or the animal treated by the target compound or the enzymolysis product of the protein with a capture agent; the capture agent is an antibody that specifically recognizes and binds the target compound and/or a conjugate containing the target compound.

2. The method of identifying a target of a compound of claim 1, wherein the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody;

preferably, the genetically engineered antibody comprises: chimeric antibodies, small molecule antibodies, bispecific antibodies, antibody conjugates, and antibody fusion proteins.

3. The method of identifying a target of a compound according to claim 2, wherein the polyclonal antibody is prepared by: immunizing a host animal with an antigen containing the target compound structure, and extracting and separating the polyclonal antibody from serum or ascites of the immunized host animal;

preferably, the host animal includes any one of a mouse, a rabbit, a goat, a monkey, and an alpaca;

preferably, the preparation of the polyclonal antibody further comprises: purifying the serum or ascites fluid to obtain the polyclonal antibody;

preferably, the purification means comprises: at least one of ultracentrifugation, salting-out precipitation, gel chromatography, ion exchange chromatography and affinity chromatography, preferably affinity chromatography.

4. The method of claim 3, wherein the antigen comprising the structure of the compound of interest is: the target compound modified by a polypeptide or a polypeptide library and coupled with a carrier protein;

preferably, the polypeptide sequence is: 5-20 amino acid sequences around the known binding site of the target compound;

preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine;

preferably, the carrier protein is selected from at least one of keyhole limpet hemocyanin, bovine serum albumin, and chicken egg albumin.

5. The method for identifying a target of a compound according to any one of claims 1 to 4, wherein the target compound is a compound having a property of covalently binding to an amino acid;

preferably, the target compound refers to a compound having a property of covalently binding to a linking group on the amino acid;

preferably, the linking group is selected from at least one of a mercapto group, a hydroxyl group, an amino group and a carboxyl group;

preferably, the amino acid is selected from: at least one of cysteine, serine, threonine, tyrosine, lysine, aspartic acid and glutamic acid.

6. The method of identifying a target of a compound of claim 5, wherein the compound of interest comprises at least one of the following small molecules:

(1) small molecules containing allylic, propargylic, cyano, vinylsulfonyl, thiosemicarbazone, ketone, quinone and/or propylene oxide groups and capable of at least one of the following reactions: performing addition reaction with cysteine sulfydryl, serine side chain hydroxyl, threonine side chain hydroxyl or tyrosine side chain phenolic hydroxyl, and performing substitution reaction with lysine side chain amino;

(2) small molecules containing haloketone, thiocyanic acid, alkyne and/or cyano groups, and capable of performing substitution reaction with sulfydryl;

(3) a small molecule containing a sulfhydryl group and capable of forming a disulfide bond with cysteine;

(4) a compound containing a glycosyl or an aglycone, which can form a glycosidic bond with a serine side chain hydroxyl group, a threonine side chain hydroxyl group or a tyrosine side chain hydroxyl group;

(5) the compound contains a propylene oxide structure and can perform ring-opening addition reaction with cysteine, lysine, aspartic acid or glutamic acid.

7. The method of identifying a target of a compound of claim 4, wherein the compound of interest is ibrutinib;

preferably, the capture agent is a polyclonal antibody specifically recognizing and binding the ibrutinib and/or proteins and polypeptides containing the ibrutinib structure;

preferably, the polypeptide sequence is: E-XXXXXXX-C (ibrutinib) -XXXXXXX-CONH₂；

Preferably, X is 18 natural amino acids other than E and C).

8. The method of identifying a target of a compound of claim 1, wherein after incubation, the method comprises detecting a capture of the capture agent;

preferably, the detection of the capture objects is identification of the capture objects using tandem mass spectrometry.

9. A method for detecting the interaction between a compound and a target is characterized in that a cell or animal protein treated by a target compound or an enzymolysis product of the protein is incubated with a capture agent; the capture agent is an antibody that can specifically recognize and bind to the target compound and/or a conjugate containing the target compound;

preferably, the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody;

preferably, the polyclonal antibody is prepared by: immunizing a host animal with an antigen containing the target compound structure, and extracting and separating the polyclonal antibody from serum or ascites of the immunized host animal;

preferably, the antigen comprising the structure of the target compound is: the target compound modified by a polypeptide or a polypeptide library and coupled with a carrier protein;

preferably, the polypeptide sequence is: 5-20 amino acid sequences around the known binding site of the target compound;

preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine;

preferably, the target compound refers to a compound having a property of covalently binding to an amino acid;

preferably, the target compound refers to a compound having a property of covalently binding to a linking group on the amino acid;

preferably, the source of the protein is selected from at least one of: a protein of a tissue or body fluid of an animal, or an enzymatic hydrolysate thereof; secreted proteins of cells or enzymatic products thereof; and a cellular protein or enzymatic hydrolysate thereof;

preferably, after incubation, the detection method comprises detection of a capture of the capture agent by an enzyme linked immunosorbent assay.

10. A method for evaluating the efficacy of a compound, comprising: incubating the protein or protein enzymolysis product solution of the animal or cell treated by the target compound with a capture agent, and detecting the combination efficiency of the capture agent and the target compound;

the capture agent is an antibody that can specifically recognize and bind to the conjugate of the target compound;

preferably, the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody;

preferably, the polyclonal antibody is prepared by: immunizing a host animal with an antigen containing the target compound structure, and extracting and separating the polyclonal antibody from serum or ascites of the immunized host animal;

preferably, the antigen comprising the structure of the target compound is: the target compound modified by a polypeptide or a polypeptide library and coupled with a carrier protein;

preferably, the polypeptide sequence is: 5-20 amino acid sequences around the known binding site of the target compound;

preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine;

preferably, the target compound refers to a compound having a property of covalently binding to an amino acid;

preferably, the target compound refers to a compound having a property of covalently binding to a linking group on the amino acid;

preferably, the source of the protein is selected from at least one of: a protein of a tissue or body fluid of an animal, or an enzymatic hydrolysate thereof; secreted proteins of cells or enzymatic products thereof; and a cellular protein or enzymatic hydrolysate thereof;

preferably, after incubation, the evaluation method comprises detection of a capture of the capture agent by an enzyme linked immunosorbent assay;

preferably, the binding efficiency of the conjugate of the capture agent and the target compound is detected by enzyme-linked immunosorbent assay.

Technical Field

The invention relates to the technical field of drug target identification, in particular to a method for identifying a target of a compound, a method for detecting interaction between the compound and the target and a method for evaluating drug effect of the compound.

Background

Most drugs affect and change the function of the human body by acting on the target on organs, tissues and cells, and produce pharmacological effects. Due to the wide variety of the structure types of the medicines, the medicine has a plurality of action targets.

The action target of the drug not only provides important information and an approach for revealing the action of the drug, but also has important effects on development and development of new drugs, establishment of screening models and discovery of lead compounds. Once the action target of the medicine is known and mastered, the approach point of new medicine development can be obtained.

Although the action target of the drug becomes an important support for reasonable drug design, the composition and function of the human body are very complex and are regulated by various factors, a plurality of natural barriers and various balances exist, and for a certain specific function, under certain conditions, a plurality of targets can be combined to play a role and also undergo pharmacokinetic processes such as absorption, rotation, distribution, metabolism and the like.

Therefore, how to identify the target of the drug faces a great challenge.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for identifying a target of a compound, a method for detecting the interaction between the compound and the target and a method for evaluating the efficacy of the compound. The identification method does not need to modify the small molecule compound in the process of identifying the compound target spot, and can truly and effectively detect the target spot of the target compound in cells and/or tissues.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a method of identifying a target of a compound, comprising:

incubating the protein of the cell or the animal treated by the target compound or the enzymolysis product of the protein with a capture agent; the capture agent is an antibody that specifically recognizes and binds the target compound and/or a conjugate containing the target compound.

The protein or the solution of the proteolysis product is incubated with a capture agent, a target compound and/or a conjugate containing the target compound in the protein solution can be specifically obtained or enriched, and whether the target compound in the protein solution has the bound target protein and the quantity and the type of the bound target protein or not is identified through mass spectrometry. The identification method is based on the specific reaction of the antigen and the antibody, does not need to modify the structure of the target compound, and can directly and effectively enrich and identify the target of the target compound.

The protein or proteolytic solution treated with the target compound is: treating a biological sample by using the target compound, and extracting the obtained protein solution from the biological sample, or obtaining a proteolysis solution after enzymolysis. In addition, the biological sample may be a non-human animal body or may be a cell. If the biological sample is a non-human animal body, the target compound is administered to the non-human animal body by injection or intragastric administration, and then the protein solution is extracted from the non-human animal body from the tissue (such as intestine, stomach, lung, heart, etc.) or body fluid (such as blood, urine, semen, etc.) to be identified. And if the cells are the target compounds, directly incubating the target compounds and the cells together, and extracting the protein solution from the cells, or performing enzymolysis to obtain a proteolysis solution. The protein solution processed by the target compound can be a protein solution obtained by directly incubating the target compound and protein or a proteolysis solution obtained by enzymolysis.

In alternative embodiments, the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody.

Preferably, the genetically engineered antibody comprises: chimeric antibodies, small molecule antibodies, bispecific antibodies, antibody conjugates, and antibody fusion proteins.

In alternative embodiments, the polyclonal antibody is prepared by: immunizing a host animal with the antigen containing the target compound structure, and extracting and separating the obtained polyclonal antibody from serum or ascites of the immunized host animal.

Preferably, the host animal includes any one of a mouse, a rabbit, a goat, a monkey, and an alpaca;

preferably, the preparation of the polyclonal antibody further comprises: purifying the serum or ascites fluid to obtain the polyclonal antibody;

preferably, the purification means comprises: at least one of ultracentrifugation, salting-out precipitation, gel chromatography, ion exchange chromatography and affinity chromatography, preferably affinity chromatography.

In an alternative embodiment, the antigen containing the structure of the target compound is: the target compound is modified by a polypeptide or a polypeptide library and coupled with a carrier protein.

Preferably, the polypeptide sequence is: the target compound has 5-20 amino acid sequences around the known binding site.

Preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine.

Preferably, the carrier protein is selected from at least one of keyhole limpet hemocyanin, bovine serum albumin, and chicken egg albumin.

In alternative embodiments, the compound of interest refers to a compound having the property of covalently binding to an amino acid;

preferably, the target compound refers to a compound having a property of covalently binding to a linking group on the amino acid;

preferably, the linking group is selected from at least one of a mercapto group, a hydroxyl group, an amino group and a carboxyl group;

preferably, the amino acid is selected from: at least one of cysteine, serine, threonine, tyrosine, lysine, aspartic acid and glutamic acid.

In alternative embodiments, the target compound comprises at least one of the following small molecules:

(1) small molecules containing allylic, propargylic, cyano, vinylsulfonyl, thiosemicarbazone, ketone, quinone and/or propylene oxide groups and capable of at least one of the following reactions: performing addition reaction with cysteine sulfydryl, serine side chain hydroxyl, threonine side chain hydroxyl or tyrosine side chain hydroxyl, and performing substitution reaction with lysine side chain amino;

(2) small molecules containing haloketone, thiocyanic acid, alkyne and/or cyano groups, and capable of performing substitution reaction with sulfydryl;

(3) a small molecule containing a sulfhydryl group and capable of forming a disulfide bond with cysteine;

(4) a compound containing a glycosyl or an aglycone, which can form a glycosidic bond with a serine side chain hydroxyl group, a threonine side chain hydroxyl group or a tyrosine side chain hydroxyl group;

(5) the compound contains a propylene oxide structure and can perform ring-opening addition reaction with aspartic acid or glutamic acid.

In alternative embodiments, the target compound is ibrutinib;

preferably, the capture agent is a polyclonal antibody that specifically recognizes and binds to the ibrutinib and/or proteins and polypeptides containing an ibrutinib structure.

Preferably, the polypeptide sequence is: E-XXXXXXX-C (ibrutinib) -XXXXXXX-CONH₂(X is selected from 18 natural amino acids except cysteine and lysine).

In an alternative embodiment, after incubation of the protein or proteolytic solution with a capture agent, the identification method comprises detecting capture of the capture agent;

preferably, the detection of the capture objects comprises: including detection of protein levels or detection of polypeptide levels;

preferably, the detection of the capture objects is identification of the capture objects using tandem mass spectrometry.

In a second aspect, the embodiments of the present invention provide a method for detecting the interaction between a compound and a target, in which a cell or animal protein treated with a target compound or an enzymatic hydrolysate of the protein is incubated with a capture agent; the capture agent is an antibody that specifically recognizes and binds the target compound and/or a conjugate containing the target compound. The detection method can directly and effectively identify the action between the target compound and the specific target protein thereof without modifying the structure of the compound.

Preferably, the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody;

preferably, the polyclonal antibody is prepared by: immunizing a host animal with an antigen containing the target compound structure, and extracting and separating the polyclonal antibody from serum or ascites of the immunized host animal;

preferably, the antigen comprising the structure of the target compound is: the target compound modified by a polypeptide or a polypeptide library and coupled with a carrier protein;

preferably, the polypeptide sequence is: the target compound has 5-20 amino acid sequences around the known binding site. So as to obtain the polyclonal antibody, and specifically recognizing a target compound to a specific protein modification site.

Preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine;

preferably, the target compound refers to a compound having a property of covalently binding to an amino acid;

preferably, the target compound refers to a compound having a property of covalently binding to a linking group on the amino acid;

preferably, the source of the protein is selected from at least one of: a protein of a tissue or body fluid of an animal, or an enzymatic hydrolysate thereof; secreted proteins of cells or enzymatic products thereof; and a cellular protein or enzymatic hydrolysate thereof;

preferably, after incubation, the detection method comprises detection of a capture of the capture agent by an enzyme linked immunosorbent assay.

In a third aspect, embodiments of the present invention provide a method for evaluating the efficacy of a compound, comprising: incubating the protein or protein enzymolysis product solution of the animal or cell treated by the target compound with a capture agent, and detecting the combination efficiency of the capture agent and the target compound;

the capture agent is an antibody that specifically recognizes and binds to the conjugate of the target compound.

The evaluation method can directly determine the bonding strength of the target compound and the target in a specific species, intuitively and fairly evaluate the drug effect of the compound, and provide a new direction for researching the action mechanism of the compound drug.

Preferably, the antibody comprises: at least one of a monoclonal antibody, a polyclonal antibody and a genetically engineered antibody.

Preferably, the polyclonal antibody is prepared by: immunizing a host animal with the antigen containing the target compound structure, and extracting and separating the obtained polyclonal antibody from serum or ascites of the immunized host animal.

Preferably, the antigen comprising the structure of the target compound is: the target compound is modified by a polypeptide or a polypeptide library and coupled with a carrier protein.

Preferably, the polypeptide sequence is: the target compound has 5-20 amino acid sequences around the known binding site. So as to obtain the polyclonal antibody, and specifically recognizing a target compound to a specific protein modification site.

Preferably, the polypeptide library sequence is represented by formula 1:

formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine.

Preferably, the target compound refers to a compound having a property of covalently binding to an amino acid.

Preferably, the target compound is a compound having a property of covalently binding to a linking group on the amino acid.

Preferably, the source of the protein is selected from at least one of: a protein of a tissue or body fluid of an animal, or an enzymatic hydrolysate thereof; secreted proteins of cells or enzymatic products thereof; and a cellular protein or an enzymatic hydrolysate thereof.

Preferably, after incubation, the evaluation method comprises detection of a capture of the capture agent by an enzyme linked immunosorbent assay.

Preferably, the binding efficiency of the conjugate of the capture agent and the target compound is detected by enzyme-linked immunosorbent assay.

The specific information and effects of the detection method and the evaluation method provided by the present application, such as the preparation of the target compound and the antibody, are the same as those described in the above identification method, and are not described herein again.

The invention has the following beneficial effects:

the embodiment of the invention provides a method for identifying a target of a compound, which comprises the steps of incubating a cell or animal protein treated by a target compound or a proteolysis product of the cell or animal protein with a capture agent; the capture agent is an antibody that specifically recognizes and binds the target compound and/or a conjugate containing the target compound. The identification method specifically acquires or enriches a target compound and/or a conjugate containing the target compound in a protein or proteolysis product solution by adopting a capture agent, and identifies whether the target compound in the protein or proteolysis product solution has a combined target and the number and the type of the combined target by mass spectrometry.

In addition, the embodiment of the invention also provides a detection method for the interaction between the compound and the target and an evaluation method for the drug effect of the compound, the detection method can effectively and rapidly identify the interaction between the compound and the specific target, and the evaluation method can intuitively and fairly evaluate the drug effect of the compound and provide a new direction for researching the action mechanism of the compound drug.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of evaluating the specificity of a conjugate of an antibody in test example 1, which binds to a target compound;

FIG. 2 is a graph showing the relationship between the concentration of a binding protein and the concentration of a drug in SU-DHL-2 cells in test example 1;

FIG. 3 is a mass MS2 spectrum of the binding protein in test example 1;

FIG. 4 shows the Western blot analysis of ibrutinib-binding protein after treating cells with HCT116 gradient in Experimental example 2;

FIG. 5 shows the results of immunofluorescence assay in cells with gradient of ibrutinib concentration in Experimental example 2;

FIG. 6 shows the mass spectrometric detection of ibrutinib-binding proteins from HCT116 cells treated with different ibrutinib concentrations in Experimental example 2;

FIG. 7 is a graph comparing the results of treating Ebrutinib binding protein in SU-DHL-2 and HCT116 cells with different concentrations of Ebrutinib in Experimental example 2;

FIG. 8 is a graph showing the results of analysis of protein-protein interaction of ibrutinib-binding protein in HCT116 cells in Experimental example 2;

FIG. 9 shows the Western blot detection result of BTK of known target protein of ibrutinib in Experimental example 3;

FIG. 10 shows the sequence of ibrutinib-binding protein in different mouse tissues in Experimental example 4;

FIG. 11 shows the analysis of the conservation of the binding site of ibrutinib in the TAP1 protein in Experimental example 4;

FIG. 12 is a three-dimensional structure simulation diagram of the combination of ibrutinib and DSTN in experimental example 4;

FIG. 13 is a three-dimensional structure simulation of the interaction of ibrutinib with surrounding amino acids in Experimental example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.