CN110658314B

CN110658314B - Method for identifying target of compound, method for detecting interaction between compound and target, and method for evaluating drug effect of compound

Info

Publication number: CN110658314B
Application number: CN201910967979.2A
Authority: CN
Inventors: 戴伦治; 王秀轩; 王新媛
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-06-29
Anticipated expiration: 2039-10-12
Also published as: CN110658314A

Abstract

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.

Description

Method for identifying target of compound, method for detecting interaction between compound and target, and method for evaluating drug effect of compound

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 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 library sequence is represented by formula 1:

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.

Example 1

This example provides a method of identifying a target for a compound, comprising the steps of:

1) preparation of antigen:

contacting the target compound with a polypeptide library (EXXXXXCXXXXXXXX-CONH)₂) Coupling Cys residues to obtain target compound modified polypeptide library (hapten), and modifying the target compoundCoupling the N end of the polypeptide of the decorated polypeptide library with lysine of carrier protein to obtain the antigen containing the target compound structure, wherein the carrier protein is BSA or KLH.

2) Preparation of antibody:

immunizing a rabbit with the antigen prepared in the step 1) of the step 1, and extracting, separating and purifying the obtained polyclonal antibody from serum or ascites of the immunized host animal;

3) target compound treatment of cells (or animals):

the solution in which the target compound is dissolved is mixed with the cells, and then the protein solution in the collected cells is lysed to determine the protein concentration in the cell fluid.

4) Detecting binding proteins (targets) of a compound of interest in a cell:

purifying the protein solution sample obtained in the step 3), purifying the protein solution sample, and performing LC-MS/MS analysis.

The mass spectral data were further analyzed using software such as MaxQuant and Mascot.

Example 2

The same as in example 1, except that in 1) the step of preparing the antigen: example 2 conjugation of a compound of interest directly to a carrier protein, omitting the polypeptide modification step.

Example 3

The same as in example 1, except that in 1) the step of preparing the antigen: the target compound modified polypeptide is 5-20 amino acid sequences around the known binding site of the target compound, so as to obtain the polyclonal antibody, and the polyclonal antibody specifically recognizes the target compound to a specific protein modification site.

Test example 1

The identification method provided in example 1 was used to identify binding proteins for ibrutinib in SU-DHL-2 cell lines, and specifically included the following steps.

1) Preparation of antigen (ibrutinib):

synthesis of a library of specific polypeptides (EXXXXXCXXXXXXXX-CONH)₂X is selected from 18 natural amino acids except cysteine and lysine), dissolving Ibrut with DMFTinib was reacted with the specific polypeptide library in borate-borax buffer at pH 9 for 24h at room temperature. After the reaction is finished, concentrating to remove the solvent, redissolving the polypeptide library by PBS, centrifuging to remove insoluble substances, coupling with carrier protein KLH, ultrafiltering, measuring the concentration, and subpackaging and storing the antigen for later use.

2) Immunizing a host animal:

feeding female New Zealand white rabbits (1.5 kg), injecting the prepared antigen subcutaneously, immunizing 1 mg/rabbit for the first time, immunizing 0.5 mg/rabbit for the subsequent time, and taking arterial blood after 4 times of total immunization.

3) Antibody purification:

the whole rabbit blood was allowed to stand overnight at 4 ℃ and centrifuged at 4000rpm at 4 ℃ for 15min, and the supernatant (serum) was collected. 7mL of serum was mixed with 1:1 of 1 XPBS solution (formulation: 8.0g/L sodium chloride, 0.2g/L potassium chloride, 2.72g/L disodium hydrogenphosphate heptahydrate, and 0.245g/L potassium dihydrogenphosphate), and Protein A immobilized beads were added thereto to react at room temperature for 2 hours. Washing with low-salt, high-salt and PBS alternately to remove foreign protein. Elution with 100mM glycine (PH 2.8) gave total IgG, PH adjusted to neutral with 1M Tris PH 8.8, and washed by PBS ultrafiltration for use.

Synthesizing a compound Cys-ibrutinib, and reacting the Cys-ibrutinib with NHS activated beads to prepare the affinity column. The resulting total IgG was added to the column and incubated overnight. The contaminating proteins were removed by alternate washing with low-salt, high-salt, 1 × PBS. The antibody that specifically binds to and recognizes ibrutinib was obtained by elution with 100mM glycine (PH 2.8), ultrafiltered with PBS, added with sodium azide and stored in a-20 o freezer.

4) Antibody specificity detection:

after dissolving 4mg of BSA in double distilled water, the solution was dissolved in 100mM TEAB, and TCEP was added thereto to give a final concentration of 20mM, and the solution was reduced at 56 ℃ for 1 hour. Dividing into two parts, and storing one part at-20 deg.C for use (control). A portion was added with 2mg of sample (ibrutinib), reacted at room temperature for 16h, and concentrated to remove the solvent.

As shown in FIG. 1, the antibody prepared by dissolving samples in 1 × loading buffer (formulation: 10% glycerol, 2% SDS, 1% β -mercaptoethanol, 5% 1M Tris-HCl, pH 6.8) to a final concentration of 0.5mg/mL and detecting the antibody (Anti-C-ibrutinib) by Western blot (Western blot assay) specifically recognizes ibrutinib-modified bovine serum albumin but not unmodified bovine serum albumin.

5) Ibrutinib treatment of SU-DHL-2 cell line:

ibrutinib was dissolved in DMSO to prepare 10mM stock, and SU-DHL-2 cells were treated with 0nM, 0.5nM, 0.1. mu.M, 1. mu.M and 10. mu.M, respectively, for 6 h. Treated SU-DHL-2 cells were harvested, washed twice with 1 XPBS, centrifuged and the supernatant removed.

To the cell fluid, 1mL of RIPA lysate (formulation: NP-401%, sodium deoxycholate 0.5%, sodium chloride 150mM, Tris (pH 7.5)50mM, nicotinamide 25mM, sodium butyrate 10 mM; 1 XProteinase inhibitor (cocktail), 1 XPatase inhibitor A solution, 1 XPatase inhibitor B solution) was added, and the cell fluid was ultrasonically lysed in ice water, centrifuged at 2000g at low temperature for 30min, and the supernatant was collected and the protein concentration was measured by the Bradford method, and diluted with the lysate to 2 mg/mL.

6) Detecting binding protein of ibrutinib in SU-DHL-2:

1mg protein sample was taken at each concentration, 4 volumes of methanol, 1 volume of chloroform and 3 volumes of water were added, respectively, and vortex centrifuged (10000g, 10 min). After centrifugation, the liquid is divided into three layers, namely an aqueous layer, a protein layer and a chloroform layer. The upper methanol and water mixed phase was gently removed, and to each of the 6 groups of samples, 4 volumes of methanol was added, vortexed and centrifuged (20000g, 10min), the supernatant was gently removed, and the protein sample was evaporated at room temperature.

The protein samples were dissolved with 400. mu.L of 50mM ammonium bicarbonate, and 60. mu.g of pancreatin was added, respectively, and the mixture was subjected to enzymatic hydrolysis at 37 ℃ overnight.

100mM DTT was added to the reaction mixture to give a final concentration of 5mM, and the mixture was reduced at 56 ℃ for 1 hour. After the samples were cooled to room temperature, 500mM iodoacetamide solutions (ready for preparation) were added to the samples, respectively, to a final concentration of 15mM, and shaken in the dark at room temperature for 30 min. 1M cysteine was added to give a final concentration of 20mM, and the reaction was carried out at room temperature for 1 hour. And (4) carrying out secondary enzymolysis, supplementing 30 mu g of pancreatin to each tube, and carrying out enzymolysis for 4 hours at 37 ℃. Decocting at 95 deg.C for 2min, and inactivating. 20000g of the polypeptide obtained after the second enzymolysis is centrifuged at 4 ℃ for 10min, the supernatant is taken and 10 XPBS is added to make the final concentration 1 XPBS. Incubate at 4 ℃ for 4h on a rotary shaker. Taking out the polypeptide, centrifuging at 4 deg.C and 20000g for 10min, and collecting the polypeptide supernatant.

After washing 40. mu.L of Protein A magnetic beads with 1ml of PBS 4 times, 50. mu.L of the antibody specific to ibrutinib was added and incubated at 4 ℃ for 4 hours, and the Protein A magnetic beads were bound to the antibody. The antibody-bound Protein A magnetic beads were washed 4 times with 1ml PBS, and the polypeptide supernatant was added and incubated overnight at 4 ℃. Washing with NETN for 2 times, washing with ETN for 1 time, and washing with water for 1 time to remove non-specific binding protein. Bound proteins were eluted with 0.1% TFA, concentrated to dryness, subjected to Zip-tip C18 column desalting, respectively, and then to LC-MS/MS analysis. The mass spectral data were further analyzed using software such as MaxQuant and Mascot.

Through data processing and analysis, 8 binding proteins are identified by the method, wherein 1 binding protein is 0.1 mu M, 3 binding proteins is 1 mu M, 8 binding sites (targets) are 10 mu M, the number of the binding sites is in positive correlation with the concentration of the drug, the specific result is shown in figure 2, and the mass spectrometry detection result refers to figure 3. The results show that the targets HMOX2, PCBP3, and ALG3 can be bound by relatively low intracellular concentrations of ibrutinib, suggesting that these proteins may be more reliable targets of ibrutinib in SU-DHL-2 cells.

Test example 2

The method of identification provided in example 1 was used to identify an ibrutinib-binding protein (target) in HCT116, and test example 2 was identified in a manner substantially identical to test example 1, except that the SU-DHL-2 cell line was replaced with the HCT116 cell line.

In step 4) of test example 2, HCT116 cells treated with increasing concentrations of ibrutinib (0nM, 0.5nM, 0.1. mu.M, 1. mu.M and 10. mu.M) were subjected to Western blot analysis and intracellular immunofluorescence analysis of the protein solution of the cells, and the Western blot results shown in FIG. 4 showed an upregulation of the immune signal. Referring to fig. 5, the results of the immunofluorescence analysis in the cells show that the antibody against ibrutinib has good specificity.

In step 6) of experimental example 2, 84 targets of ibrutinib were identified, see fig. 6. Among them, the target CKAP4 was detected in ibrutinib-treated cells at different concentrations, indicating a strong interaction between CKAP4 and ibrutinib. CKAP4 is a DKK1 receptor that regulates dickkopf1(DKK1) signaling, playing an important role in tumor cell proliferation.

In conjunction with test example 1, the identification methods provided herein found targets from both the SU-DHL-2 and HCT116 cell lines: HMOX2, PCBP3, ALG3, SEC61B (transporter) and NT5DC1, see fig. 7.

The inventors further performed protein-protein interaction analysis of 84 binding proteins of ibrutinib identified in HCT116 cell line, see figure 8. The targets of ibrutinib in SU-DHL-2 cells analyzed by the website https:// string-db.org are proteins such as HMOX2, PCBP3, ALG3, and NT5DC1, and few other proteins interact with these targets, indicating that these targets are direct targets of ibrutinib in SU-DHL-2.

Test example 3

The identification of ibrutinib-binding proteins (targets) in HCT116 was performed using the identification method provided in example 1, which is substantially the same as in Experimental example 1, except that the ibrutinib-treated SU-DHL-2 cell line was replaced with ibrutinib-treated BTK wild-type cells and BTK^C418SA mutant cell. BTK is a known target of ibrutinib.

Western blot assay results referring to FIG. 9, it is shown that anti-ibrutinib antibodies were detected only in ibrutinib-treated BTK wild-type cells and in BTK^C481SThe antibody can not be detected in mutant cells, and the ibrutinib specific antibody has specificity and reliability, namely the capture agent provided by the embodiment of the application has accuracy.

Test example 4

Identification of ibrutinib binding proteins (targets) in animal models.

1) Preparation of antigen (same as in test example 1);

2) immunization of host animals (same as test example 1);

3) antibody purification (same as in test example 1);

4) antibody-specific detection (same as test example 1);

5) breeding the mice: dividing mice into two groups with equal amount, performing gastric lavage (60mg/kg) on one group, performing gastric lavage on the other group with physiological saline with equal volume, collecting nine tissues including heart, liver, spleen, lung, kidney, stomach, small intestine, brain and colon after 6h, washing blood with physiological saline, quickly freezing with liquid nitrogen, and storing at-80 deg.C;

6) extracting proteins of each tissue of the mouse;

nine tissue samples of each mouse, 100mg, were taken, 1mL of RIPA lysate was added, the tissues were disrupted using a tissue homogenizer, sonicated, centrifuged at high speed and low temperature, the supernatant was taken, and the protein concentration was measured by the Bradford method and diluted with lysate to a protein concentration of 2 mg/mL.

7) Detection of Ibrutinib targets (binding proteins) in various tissues

A3 mg protein sample was taken from each tissue, added with 4 volumes methanol, 1 volume chloroform and 3 volumes water, respectively, and vortexed (10000g, 10 min). After centrifugation, the liquid is divided into three layers, namely an aqueous layer, a protein layer and a chloroform layer. The upper methanol and water mixed phase was gently removed, and to 6 groups of samples, 4 volumes of methanol were added, vortexed and centrifuged (20000g, 10min), the supernatant was gently removed, and the protein samples were evaporated at room temperature.

100mM DTT was added to the reaction mixture to give a final concentration of 5mM, and the mixture was reduced at 56 ℃ for 1 hour. After the samples were cooled to room temperature, 500mM iodoacetamide solutions (ready for preparation) were added to the samples, respectively, to a final concentration of 15mM, and shaken in the dark at room temperature for 30 min. 1M cysteine was added to give a final concentration of 20mM, and the reaction was carried out at room temperature for 1 hour.

And (4) carrying out secondary enzymolysis, supplementing 30 mu g of pancreatin to each tube, and carrying out enzymolysis for 4 hours at 37 ℃. Decocting at 95 deg.C for 2min, inactivating to obtain polypeptide, centrifuging polypeptide 20000g at 4 deg.C for 10min, collecting supernatant, and adding 10 × PBS to give final concentration of 1 × PBS. Incubate at 4 ℃ for 4h on a rotary shaker. Taking out the polypeptide, centrifuging at 4 deg.C and 20000g for 10min, and collecting the polypeptide supernatant.

And (3) incubating the Protein A magnetic beads and the ibrutinib specific antibody for 4h at 4 ℃, combining the Protein A magnetic beads with the antibody, then adding the polypeptide supernatant, and incubating overnight at 4 ℃. And sequentially washing with NETN, ETN and water to remove non-specific binding protein. Bound proteins were eluted with 0.1% TFA, concentrated to dryness, subjected to Zip-tip C18 column desalting, and analyzed by LC-MS/MS, respectively.

By data processing analysis, the method detected no binding protein in heart, lung, kidney and brain tissue, and detected binding protein in stomach, small intestine, colon, liver and spleen, and identified 9 binding proteins in total, the results are shown in fig. 10.

From the above results, no binding site (target) for ibrutinib was detected in mouse lung, kidney and brain, and the targets of Ses61b and destin (dstn) were found in both human cells and mouse models in all binding proteins identified by mass spectrometry for ibrutinib. DSTN is an actin-binding protein, and loss of expression of DSTN is a biomarker of many cancers, so that it is speculated that side effects caused by ibrutinib in many organs may be due to the inhibition of DSTN.

Detecting the interaction of ibrutinib with a target:

the three-dimensional structure of Desrin (PDB ID:1AK7) was downloaded from the RCSB protein database (www.rcsb.org). The 3D structure of ibrutinib is generated by Discovery Studio v3.1 software. And carrying out Covalent Docking calculation on the two structures in Schrodinger suite 2018 software according to a Glide equivalent Docking protocol. The resulting images were processed with PyMoL v1.8 software (fig. 12 and 13), and the three-dimensional structure showed good surface bonding of ibrutinib to DSTN, see fig. 12. According to computer modeling, referring to FIG. 13, there may be a π -cation interaction between ARG13 and ibrutinib, and a hydrogen bond between Asp17 and ibrutinib.

Tap1 was detected in the stomach, small intestine, colon, liver and spleen, and Ses61b was detected in the stomach consistent with that in the SU-DHL-2 cell line, the mass spectrometric results are shown in fig. 11.

Antigenic peptide transporter 1(TAP1) is a member of the ATP-binding cassette (ABC) transporter family. The combination of ibrutinib and TAP1 is found in Cys150 of 5 organs such as the large intestine, the small intestine, the stomach, the liver, the spleen and the like of a mouse, and the strong interaction between the ibrutinib and TAP1 is shown. Sequence fasta files of various species of TAP1 were downloaded in uniprot and sequence alignment analysis by MEGA software found that Cys150 was not a conserved site between human and mouse. Also no target TAP1 was observed to be found in human cells, consistent with the results of the evolutionary analysis. Animal models have long played an important role in elucidating models of drug action. The observed target distribution among species explains why some drug candidates are very effective in mice, but not in humans.

Ibrutinib is an FDA-approved targeting drug for treating mantle cell lymphoma, and can be covalently bound with cysteine residues in BTK active centers, thereby inhibiting the activity thereof and inhibiting the proliferation and survival of malignant B cells.

In example 1, a human B lymphocyte cell line is selected first, and binding proteins of ibrutinib in a SU-DHL-2 cell line are identified, and the method identifies 8 binding proteins in total, wherein 0.1 white, 1 white, 3 white and 8 white 10, the number of binding sites (target points) is positively correlated with the drug concentration, and the specific result is shown in fig. 2, and the mass spectrometry detection result refers to fig. 3.

The results show that the targets HMOX2, PCBP3, and ALG3 can be bound by relatively low intracellular concentrations of ibrutinib, suggesting that these proteins may be more reliable targets of ibrutinib in SU-DHL-2 cells. To further determine the binding protein of ibrutinib, we selected a cell line HCT116 cell line unrelated to the human B lymphocyte cell line, and in example 2, 84 binding proteins of ibrutinib identified in HCT116 cell line, targets found in both SU-DHL-2 and HCT116 cell lines: HMOX2, PCBP3, ALG3, SEC61B (transporter) and NT5DC1, with fewer interacting targets, suggesting that these targets are direct targets of ibrutinib in SU-DHL-2.

Example 3 the identification method was substantially the same as in test example 1, except that the ibrutinib-treated SU-DHL-2 cell line was replaced with ibrutinib-treated BTK wild-type cells and BTKC418S mutant cells. BTK is a known target of ibrutinib. The results show that the anti-ibrutinib antibodies are only on ibrutinibIt was detected in the BTK wild-type cells treated with lurtinib, whereas in BTK^C481SThe antibody can not be detected in mutant cells, and the ibrutinib specific antibody has specificity and reliability, namely the capture agent provided by the embodiment of the application has accuracy.

In summary, the embodiments of the present invention provide a method for identifying a target of a compound,

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

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 can specifically recognize and bind to the target compound and/or a conjugate containing the target compound;

the antibody is a polyclonal antibody;

the preparation of the polyclonal antibody comprises the following steps: 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;

the antigen containing the structure of the target compound is: the target compound modified by a polypeptide library and coupled with a carrier protein; the target compound is a compound with the property of covalent binding with amino acid; the target compound is a compound with the property of covalent bonding with a connecting group on the amino acid;

the sequence of the polypeptide library is shown as formula 1:

the carrier protein is at least one of keyhole limpet hemocyanin, bovine serum albumin and chicken egg albumin;

the identification method comprises performing mass spectrometry on a capture of the capture agent.

2. The method of identifying a target of a compound according to claim 1, wherein the host animal comprises any one of a mouse, a rabbit, a goat, a monkey, and an alpaca.

3. The method of identifying a target of a compound of claim 1, wherein the preparation of the polyclonal antibody further comprises: purifying the serum or ascites to obtain the polyclonal antibody.

4. The method of identifying a target of a compound according to claim 3, wherein the purification comprises: at least one of ultracentrifugation, salting-out precipitation, gel chromatography, ion exchange chromatography and affinity chromatography.

5. The method of identifying a target of a compound according to claim 4, wherein the purification is affinity chromatography.

6. The method of identifying a target of a compound of claim 1, wherein the linking group is selected from at least one of a thiol group, a hydroxyl group, an amino group, and a carboxyl group.

7. The method of identifying a target of a compound according to claim 1, wherein said amino acid is selected from the group consisting of: at least one of cysteine, serine, threonine, tyrosine, lysine, aspartic acid and glutamic acid.

8. The method of identifying a target of a compound of claim 1, 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;

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

9. The method of identifying a target of a compound of claim 1, wherein the compound of interest is ibrutinib.

10. The method of identifying a target for a compound of claim 9, wherein the capture agent is a polyclonal antibody that specifically recognizes and binds said ibrutinib and/or proteins and polypeptides containing an ibrutinib structure.

11. The method of claim 10, wherein the capture is detected by tandem mass spectrometry.

12. 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;

the antibody is a polyclonal antibody; the preparation of the polyclonal antibody comprises the following steps: 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;

the sequence of the polypeptide library is shown as formula 1: formula 1: E-XXXXXXX-C-XXXXX-CONH₂(ii) a Wherein X is selected from 18 natural amino acids except cysteine and lysine;

the detection method comprises performing mass spectrometry on a capture of the capture agent.

13. The method of claim 12, wherein the protein is derived from at least one of the following: 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.