Method for identifying covalent target spot of antitumor drug and application of covalent target spot

Abstract

The invention provides a method for identifying a covalent target spot of an antitumor drug and application of the covalent target spot, and relates to the technical field of chemical proteomics. The method comprises the following specific steps: S1, labeling competitive active groups through a probe to obtain labeled protein; s2, performing enzymolysis and affinity enrichment on the labeled protein to obtain an enriched peptide fragment sample; s3, performing quantitative mass spectrometry on the peptide fragment sample to obtain a binding site; s4, performing pathway enrichment analysis on the binding sites to obtain covalent targets of the antitumor drugs; the antitumor drug is 4-iodo-3-nitrobenzamide, and the probe is a DBIA probe; according to the invention, the high-confidence covalent binding site of the 4-iodine-3-nitrobenzamide is systematically identified and positioned at the proteome level for the first time, and the covalent targets of the 4-iodine-3-nitrobenzamide are successfully identified as PLEC, KRI1 and COF1; the multi-target feature shows that the 4-iodine-3-nitrobenzamide realizes systematic regulation and control on tumor cells through a structure-function multi-channel integration mechanism.

Description

Technical Field

[0001] This invention relates to the field of chemical proteomics technology, specifically to a method for identifying covalent targets of antitumor drugs and its applications. Background Technology

[0002] Iniparib (chemical name: 4-iodo-3-nitrobenzamide, code name BSI-201) is an early-developed antitumor candidate compound. Its molecular formula is C7H5IN2O3, its molecular weight is 292.03 Da, and its structure contains iodine atoms, nitro groups, and amide functional groups.

[0003] This compound was initially thought to be a poly ADP-ribose polymerase (PARP) inhibitor. PARP family proteins play a crucial role in DNA damage repair, promoting the recruitment of DNA repair proteins and complex formation through catalytic ADP-ribosylation. Inhibition of PARP activity can lead to DNA repair blockage, thereby triggering a "synthetic lethality" effect in homologous recombination repair (HRR) cells, achieving selective killing of tumor cells.

[0004] Subsequent studies have shown that the bioactivity of iniparib differs significantly from that of typical PARP inhibitors. Iniparib has a very weak inhibitory effect on PARP1 / PARP2 and cannot effectively block the formation of intracellular poly(ADP-ribose) (PAR). Its cytotoxicity only occurs at higher concentrations and it lacks selectivity for BRCA / homogeneous recombination repair deficient (HR-deficient) cells. Therefore, its pharmacological mechanism urgently needs to be re-evaluated.

[0005] However, no studies have yet systematically elucidated the covalent modification profile and target specificity of iniparib at the proteomic level. Traditional target discovery methods struggle to reveal the specific covalent binding sites between drugs and proteins, thus failing to fully elucidate its multi-target mechanism of action.

[0006] Therefore, exploring the covalent targets of the anti-tumor drug Iniparib is an urgent problem to be solved. Summary of the Invention

[0007] Specifically addressing the shortcomings of existing technologies, this invention provides a method for identifying covalent targets of antitumor drugs and its applications. The specific steps of the method are as follows: S1, competitive labeling with a probe to obtain a labeled protein; S2, enzymatic digestion and affinity enrichment of the labeled protein to obtain enriched peptide samples; S3, quantitative mass spectrometry analysis of the peptide samples to obtain binding sites; S4, pathway enrichment analysis of the binding sites to obtain the covalent targets of the antitumor drug; the antitumor drug is 4-iodo-3-nitrobenzamide, and the probe is a DBIA probe. This invention is the first to systematically identify and locate high-confidence covalent binding sites of 4-iodo-3-nitrobenzamide at the proteomic level, successfully identifying PLEC, KRI1, and COF1 as covalent targets of 4-iodo-3-nitrobenzamide. This multi-target characteristic indicates that 4-iodo-3-nitrobenzamide achieves systematic regulation of tumor cells through a structure-function multi-pathway integration mechanism.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for identifying covalent targets of antitumor drugs, the specific steps of which are as follows: S1. Competitive labeling of active groups using probes yields labeled proteins; S2. The labeled protein is enzymatically digested and affinity enriched to obtain enriched peptide samples. S3. Perform quantitative mass spectrometry analysis on the peptide sample to obtain the binding sites; S4. Pathway enrichment analysis of the binding sites was performed to obtain the covalent targets of antitumor drugs; The antitumor drug is 4-iodo-3-nitrobenzamide, and the probe is a DBIA probe.

[0009] The structural formula of 4-iodo-3-nitrobenzamide is as follows:

[0010] In some specific embodiments of the present invention, the covalent target is PLEC, KRI1, and COF1.

[0011] In some specific embodiments of the present invention, the specific steps of competitive active group labeling by the probe in step S1 are as follows: 4-iodo-3-nitrobenzamide is added to breast cancer cells and incubated for 16-20 hours. After lysis, the cell supernatant is collected. DBIA lysis buffer is added to the cell supernatant and reacted in the dark for 0.5-1.5 hours. Dithiothreitol is added and reacted for 20-40 minutes. Iodoacetamide is then added and reacted in the dark for 20-40 minutes. Dithiothreitol is then added again and reacted for 10-20 minutes to obtain the labeled protein.

[0012] In some specific embodiments of the present invention, the breast cancer cells are MDA-MB-231 cells.

[0013] In some specific embodiments of the present invention, the concentration of the 4-iodo-3-nitrobenzamide is 30 μM; The cell lysis buffer consisted of 50 mM HEPES, 0.1% nuclease, 1× protease inhibitor, 2% sodium dodecyl sulfate, and 8 M urea. The DBIA lysis buffer consisted of 50 mM HEPES, 2% sodium dodecyl sulfate, 8 M urea, and 2 mM DBIA. The final concentration of added dithiothreitol is 8-12 mM, preferably 10 mM; the final concentration of added iodoacetamide is 28-32 mM, preferably 30 mM; and the final concentration of added dithiothreitol is 23-28 mM, preferably 25 mM.

[0014] In some specific embodiments of the present invention, the specific steps of enzymatic digestion and affinity enrichment in step S2 are as follows: the labeled protein is digested with enzymes to obtain peptide samples; then streptavidin magnetic beads are used to enrich the peptide samples to obtain enriched peptide samples.

[0015] In some specific embodiments of the present invention, the specific steps of the quantitative mass spectrometry analysis in step S3 are as follows: the enriched peptide sample is eluted to obtain dried peptides; the dried peptides are then subjected to LC-MS / MS acquisition, database search and data processing; then differential site screening and spectrum verification are performed to obtain the binding sites of 4-iodo-3-nitrobenzamide. The screening threshold for the differential site selection is Fold change ≥ 4 and p -value<0.05.

[0016] In some specific embodiments of the present invention, the database for data processing is UniProt-human-filtered-reviewed, the enzyme digestion method is Trypsin / P, the variable modifications are DBIA-Cys[C], Carbamidomethyl[C], Acetyl[Protein N-term] and Oxidation[M], the maximum missed digestion is 2, and the FDR is 1%.

[0017] In some specific embodiments of the present invention, the specific steps of the pathway enrichment analysis in step S4 are as follows: the target protein of the binding site is subjected to GO pathway enrichment analysis and KEGG pathway enrichment analysis, and then Fisher's exact test is used to obtain the covalent target of 4-iodo-3-nitrobenzamide. The significance threshold for the Fisher exact test is set to FDR < 0.05.

[0018] Secondly, the present invention provides the use of the method described in the first aspect for identifying covalent targets of antitumor drugs.

[0019] The beneficial effects achieved by this invention are as follows: 1. This invention is the first to systematically identify and locate high-confidence covalent binding sites of 4-iodo-3-nitrobenzamide at the proteomic level. The covalent targets of 4-iodo-3-nitrobenzamide were successfully identified as PLEC, KRI1, and COF1, which are involved in cytoskeleton and signal transduction, ribosome biosynthesis and metabolic regulation, DNA damage repair and stress response. This multi-target feature indicates that 4-iodo-3-nitrobenzamide achieves systematic regulation of tumor cells through a structure-function multi-pathway integration mechanism.

[0020] 2. The high-confidence target of 4-iodo-3-nitrobenzamide provided by this invention clarifies the covalent binding relationship between the drug and a specific site, which can directly guide the pharmacological repositioning, mechanism of action research and derivative optimization design of 4-iodo-3-nitrobenzamide, and provide molecular-level evidence for its transformation from a "PARP-like inhibitor" to a "multi-target covalent modulator".

[0021] The abbreviations for the reaction reagents mentioned in the instructions are as follows: DTT: Dithiothreitol; IAA: Iodoacetamide; Trypsin: trypsin; LysC: lysine protease; TEAB: Triethylammonium bicarbonate; SP3: Carboxyl magnetic beads. Attached Figure Description

[0022] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Volcano diagram of the protein binding site of the antitumor drug Iniparib; Figure 2 GO annotation statistics of differentially expressed proteins at Iniparib protein binding sites (level 2); Figure 3 A map showing the enrichment of GO term biological process pathways that are significantly enriched in differentially expressed proteins at the Iniparib protein binding site. Figure 4A pathway enrichment map of GO term cellular components that are significantly enriched in differentially expressed proteins at the Iniparib protein binding site. Figure 5 A functional pathway enrichment map of GO term molecules that are significantly enriched in differentially expressed proteins at the Iniparib protein binding site; Figure 6 Bubble plot showing KEGG pathway enrichment significantly enriched among differentially expressed proteins at the Iniparib protein binding site; Figure 7 Volcano diagram of covalent targets for the antitumor drug Iniparib. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1: Peptide Sample Preparation and DBIA Probe Labeling Cell line: MDA-MB-231 (human breast cancer cells); Small molecules: Iniparib (purity ≥98%), dimethyl sulfoxide (DMSO, solvent control); Probe: DBIA (Dithiothreitol-Biotin-Iodoacetamide); The Chinese name is N-(2-(2-iodoacetamido)ethyl)-6-(5-methyl-2-oxoimidazolidine-4-yl)hexamethyleneamide; Brand: ChomiX, Model: 02010001.

[0026] Reagents: Dithiothreitol (DTT), Iodoacetamide (IAA), Trypsin, Lysine protease (LysC), Triethylammonium bicarbonate (TEAB), Acetonitrile, Formic acid, Streptavidin beads, Carboxyl magnetic beads (SP3), etc.

[0027] (1) Cell culture and drug treatment Human breast cancer cells MDA-MB-231 were seeded in 10 cm culture dishes and cultured in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and 5% CO2. When the cell density reached approximately 90% confluence, the medium was discarded, and the cells were washed once with phosphate-buffered saline (PBS). Subsequently, 10 mL of DMEM medium containing 30 μM iniparib (Iniparib group) and 10 mL of DMEM medium containing an equal volume of DMSO (DMSO group, blank control group) were added to the culture dishes, and the cells were incubated for another 18 hours at 37°C and 5% CO2. After incubation, the cells were washed twice with PBS. The cells were collected, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0028] (2) Sample pyrolysis Remove cell samples stored at -80°C and thaw slowly on ice. Add 520 μL of cell lysis buffer to each cell sample. Sonicate at 20°C. Centrifuge the cell lysis buffer at 20,000 g for 30 minutes at 25°C, collect the supernatant, determine the protein concentration using a BCA assay kit, and dilute to 2 mg / mL for later use.

[0029] The cell lysis buffer consisted of 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, pH = 8.5), 0.1% nuclease, 1× protease inhibitor, 2% sodium dodecyl sulfate (SDS), and 8 M urea.

[0030] (3) Probe marking Take 50 μL of protein sample and place it in an EP tube. Add 50 μL of lysis buffer containing 2 mM DBIA and react at room temperature in the dark for 1 hour. Add DTT (final concentration 10 mM) and react at 37°C for 30 minutes. Add IAA (final concentration 30 mM) and react at room temperature in the dark for 30 minutes. Add DTT (final concentration 25 mM) and react at room temperature for 15 minutes to complete the denaturation, reduction and cysteine ​​blocking of the protein, and obtain the labeled protein.

[0031] The lysis buffer for DBIA consisted of 50 mM HEPES (pH 8.5), 2% SDS, 8 M urea, and 2 mM DBIA.

[0032] (4) Protein precipitation Protein was adsorbed using SP3 magnetic beads (concentration 25 μg / μL), and ethanol was added to a concentration of 50% (v / v). The mixture was reacted at 25°C for 10 minutes to precipitate the protein. The beads were then separated using a magnetic rack, the supernatant was discarded, and the mixture was washed three times with an 80% ethanol aqueous solution.

[0033] (5) Enzyme digestion Add 100 μL of TEAB (100 mM), 2 μg of Trypsin, and 2 μg of LysC to each SP3 magnetic bead sample, and incubate at 37°C for 16 hours by rotation. After enzyme digestion, elute the peptides with PBS, and collect the supernatants and combine them.

[0034] (6) Enrichment washing Take 500 μL of streptavidin magnetic beads, wash three times with PBS, and resuspend in 300 μL of PBS. Add 100 μL of the resuspended streptavidin magnetic beads to each sample, and incubate at 25 °C with rotation for 3 hours. Add another 100 μL of streptavidin magnetic beads to the sample every hour. After enrichment, wash the magnetic beads three times with 1 mL of PBS containing 1 M NaCl, and then wash the magnetic beads three times each with 1 mL of PBS and 1 mL of deionized water to obtain the enriched peptide samples.

[0035] (7) Peptide elution The peptide fragments were eluted with 50% acetonitrile-water (containing 0.1% formic acid), dried by rotary evaporation, desalted by C18, and dried again by rotary evaporation to obtain dried peptide fragments for later use.

[0036] Example 2: LC-MS / MS Acquisition and Database Search Instruments and conditions LC system: Vanquish Neo UHPLC Mass spectrometry: Q Exactive HF-X Ion source: Nanospray ESI (positive ion mode) Acquisition mode: DIA Column: Acclaim PepMap RSLC C18 (75μm×25 cm, 2μm, 100 Å) Gradient: 0 min 3%B - 5 s 7%B - 72 min 20 s 34%B - 86 min 15 s 40%B - 86 min 20 s 100%B - 95 min 100%B - stop The constant flow rate is 300 nL / min.

[0037] MS1: 350-1800 m / z, 60,000 resolution DIA-MS2: 45 windows, 30,000 resolution, 30% HCD resolution. Data Processing: Raw LC-MS / MS data were imported into Spectronaut (V18.2) software for database searching. The database used was UniProt-human-filtered-reviewed, with Trypsin / P restriction enzymes, and variable modifications including DBIA-Cys[C], Carbamidomethyl[C], Acetyl[Protein N-term], and Oxidation[M]. The maximum missed cleavage was 2, and the FDR was 1%. Two treatment groups were used: DMSO (2 biological replicates) and Iniparib (3 biological replicates), for a total of 5 data sets. .tsv files were exported for further analysis.

[0038] Example 3: Screening of Differential Sites and Spectral Verification Difference screening: After exporting the table, data with DBIA-Cys sites were filtered out, while data with missing Quantity in the DMSO group were removed. The quantitative ratio of DBIA-modified Cys sites between the DMSO and Iniparib groups (Quantity ratio_DMSO / Iniparib) was calculated, and Welch's t-test was used to calculate the significance level of the difference between the groups. p (Value). With Fold change ≥ 4 and p -value<0.05 is the screening threshold. Verify the spectra of all candidate sites and remove false positive spectra.

[0039] Results: After screening and spectral verification using the above process, 29 high-confidence Iniparib binding sites were obtained (the abundance of sites labeled by the DBIA probe was significantly higher in the DMSO group than in the Iniparib group, indicating that the site was occupied by Iniparib). The Iniparib binding site volcano map is shown below. Figure 1 As shown, its site identifiers include (but are not limited to): O00159_C625, P18031_C344, Q8N9T8_C591, P68104_C411, Q15149_C1156, Q14699_C211, Q14315_C728, Q 15149_C530, P07900_C572, P38646_C487, P0DN76_C169, P35606_C190, P62140_C244, O00232_C31, Q166 58_C226, P16152_C122, P23528_C147, Q96TA1_C512, P63261_C285, P32119_C172, Q15392_C91, P61160_ C20, O75643_C769, P14618_C326, P08238_C564, P43487_C158, Q9UBQ5_C190, P18077_C47, P00367_C376.

[0040] Example 4: Functional enrichment and domain analysis of differentially expressed proteins Considering the multi-target effects of Iniparib, and in order to fully understand the activity, functional localization, and biological processes involved in the target proteins, the inventors conducted GO / KEGG pathway enrichment analysis.

[0041] GO / KEGG analysis method: GO (CC / BP / MF) and KEGG pathway enrichment analysis was performed on 28 target proteins from 29 high-confidence target sites. Fisher's exact test was used, and the significance threshold was set to FDR<0.05. An enrichment bubble chart was drawn to show the top 10 significant items.

[0042] (1) Classification of GO function annotations GO (Gene Ontology), also known as GO term, is a classification system established to describe the biological functions of biological macromolecules. GO annotations are divided into three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The inventors statistically analyzed the number of differentially expressed proteins at the GO secondary functional annotation level. A statistical graph of GO annotations for differentially expressed proteins at the Iniparib binding site (level 2) is shown below. Figure 2 As shown.

[0043] (2) GO enrichment analysis To demonstrate the enrichment trend of differentially expressed proteins in the GO annotation pathway, Fisher's exact test was performed on the differentially expressed proteins to obtain the enrichment of differentially expressed proteins in GO terms. The significance level of the enrichment of GO terms was then assessed. p -value) to identify functional classifications and pathways where differentially expressed proteins are significantly enriched. Select significantly enriched ( p The top 10 GOterms with -value ≤ 0.05 were plotted in an enrichment bubble diagram. The enrichment results for biological process enrichment pathways (BP-enriched pathways) are shown below. Figure 3 As shown, it was significantly enriched in pathways such as positive regulation of tau-protein kinase activity, telomerase holoenzyme complex assembly, and positive regulation of lamellipodium assembly. The enrichment results of the cellular component enrichment pathway (CC-enrichment Pathway) are as follows: Figure 4 As shown, the enrichment results of molecular function-enriched pathways (MF-enriched pathways) are as follows: Figure 5 As shown.

[0044] In the GO term enrichment bubble plot, the horizontal axis represents Log2 (Fold enrichment), indicating the enrichment degree of differentially expressed proteins in GO terms. Fold enrichment = (number of differentially expressed proteins in GO terms / total number of differentially expressed proteins) / (number of proteins in GO terms / total number of proteins in all GO terms). The vertical axis represents the GO terms enriched, and the color in the plot indicates the significance of enrichment. p -value), the size of the circle indicates the number of differentially expressed proteins in the GO term.

[0045] (3) KEGG pathway enrichment analysis To identify the enrichment trend of differentially expressed proteins in the KEGG pathway, Fisher's exact test was performed on the differentially expressed proteins to obtain the enrichment status of differentially expressed proteins in the KEGG pathway. The significance level of the enrichment in the KEGG pathway was then assessed. p -value) to identify KEGG pathways with significantly enriched differentially expressed proteins. Select significantly enriched ( p For the top 10 KEGG pathways with -value ≤ 0.05, an enrichment bubble plot was plotted.

[0046] A bubble map showing significant enrichment of the KEGG pathway in differentially expressed proteins at the Iniparib binding site, as shown. Figure 6As shown in the figure, the horizontal axis of the KEGG enrichment bubble plot is Log2 (Fold enrichment), which represents the degree of enrichment of differentially expressed proteins in the KEGG pathway. Fold enrichment = (number of differentially expressed proteins in the KEGG pathway / total number of differentially expressed proteins) / (number of proteins in the KEGG pathway / total number of proteins in all KEGG pathways). The vertical axis represents the enriched KEGG pathway, and the color in the figure indicates the significance of enrichment. p -value), the size of the circle indicates the number of differentially expressed proteins in KEGG.

[0047] KEGG enrichment bubble plots showed that differentially expressed proteins were significantly enriched in pathways such as the Citrate cycle (TCA cycle), Aminoacyl−tRNA biosynthesis, and pyruvate metabolism.

[0048] The main enrichment results obtained from the GO / KEGG pathway enrichment analysis are as follows: Cellular components (CC): spliceosome, endoplasmic reticulum / Golgi-associated vesicle membrane, microvilli; Biological processes (BP): RNA processing, protein folding, pseudopodia assembly regulation, host-virus interaction; Molecular functions (MF): ATP binding, thiol transferase activity, chaperone protein binding, actin filament binding; KEGG pathways include: glycolysis / pyruvate metabolism / TCA cycle, protein processing (ER), actin cytoskeleton regulation, insulin signaling pathway, and infection / necrotizing cell death pathway.

[0049] Analysis of the domains, predicted by InterPro / InterProScan, revealed that the differentially expressed protein was rich in ATP / NAD(P) binding domains, P-loop NTP enzyme-like domains, Armadillo / HEAT-like repeats, and Winged-helix-like domains, suggesting that they are mostly catalytic and ligand binding sites and are susceptible to functional activity affected by Iniparib covalent modification.

[0050] Using the Abundance Ratio (log2) at each site and p The summary analysis using -value(-Log10) yielded the following results: Figure 7The volcano plot of covalent targets for the antitumor drug Iniparib shown reveals significant differences among all results, particularly at cysteine ​​residues 530 and 1156 of Plectin (PLEC), cysteine ​​residue 591 of KRI1 homolog (KRI1), and cysteine ​​residue 147 of the cofactor of BRCA1 (COF1). To further elucidate the mechanism of action of Iniparib, functional analysis was performed on its target proteins PLEC, KRI1, and COF1, and the specific mechanisms of action are as follows.

[0051] PLECs are high-molecular-weight cytoskeleton connective proteins responsible for integrating intermediate filaments, microfilaments, and microtubules into a structural network, maintaining cellular mechanical stability. This invention reveals that iniparib can covalently modify PLECs at Cys530 and Cys1156, interfering with the binding of PLECs to actin, thereby affecting cell migration and membrane stability. PLECs are upregulated during tumor cell invasion, modulating focal adhesion signaling and ROS balance. Therefore, iniparib modification of PLECs leads to abnormal cytoskeleton stress and inhibited motility, indicating its potential anti-metastatic mechanism.

[0052] KRI1 is a ribosomal biosynthesis regulatory protein involved in 18S rRNA precursor processing and 40S ribosomal subunit assembly. Its deficiency can induce ribosomal stress and activate the p53 pathway. Iniparib covalently modifies KRI1's Cys591, blocking its binding to complexes such as NOB1 and RCL1, interfering with ribosomal maturation, and reducing protein translation efficiency. The hypermetabolic state of tumor cells depends on active ribosome production; this result indicates that iniparib inhibits tumor cell proliferation by generating metabolic stress through the inhibition of ribosomal biosynthesis and translational activity.

[0053] COF1 is a BRCA1 complex interacting protein involved in DNA damage repair and chromosome stability maintenance. The covalent binding of iniparib at the Cys147 site weakens the binding affinity of COF1 to BRCA1, inhibiting homologous recombination repair (HRR) and reducing DNA repair efficiency. This mechanism suggests that iniparib may have a synergistic or complementary effect with PARP inhibitors, exhibiting selective sensitivity to BRCA-deficient tumors, providing a basis for their clinical retargeting.

[0054] Iniparib's key targets include the cytoskeleton and signal transduction (PLEC), ribosome biosynthesis and metabolic regulation (KRI1), and DNA damage repair and stress response (COF1). This multi-target characteristic indicates that Iniparib achieves systemic regulation of tumor cells through a structure-function multi-pathway integration mechanism, providing molecular-level evidence for its transformation from a "PARP-like inhibitor" to a "multi-target covalently modulator." High-confidence target information can directly guide the pharmacological repositioning of Iniparib, mechanism of action studies, and derivative optimization design. Clarifying the covalent binding relationship between the drug and specific sites helps optimize selectivity and affinity, and explore potential clinical indications.

[0055] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A method for identifying covalent targets of antitumor drugs, characterized in that, The specific steps of the method are as follows: S1. Competitive labeling of active groups using probes yields labeled proteins; S2. The labeled protein is enzymatically digested and affinity enriched to obtain enriched peptide samples. S3. Perform quantitative mass spectrometry analysis on the peptide sample to obtain the binding sites; S4. Pathway enrichment analysis of the binding sites was performed to obtain the covalent targets of antitumor drugs; The antitumor drug is 4-iodo-3-nitrobenzamide, and the probe is a DBIA probe.

2. The method for identifying covalent targets of antitumor drugs according to claim 1, characterized in that, The covalent targets are PLEC, KRI1, and COF1.

3. The method for identifying covalent targets of antitumor drugs according to claim 1 or 2, characterized in that, The specific steps for competitive active group labeling using the probe in step S1 are as follows: 4-iodo-3-nitrobenzamide is added to breast cancer cells and incubated for 16-20 hours. After lysis, the cell supernatant is collected. DBIA lysis buffer is added to the cell supernatant and reacted in the dark for 0.5-1.5 hours. Dithiothreitol is added and reacted for 20-40 minutes. Iodoacetamide is then added and reacted in the dark for 20-40 minutes. Dithiothreitol is then added again and reacted for 10-20 minutes to obtain the labeled protein.

4. The method for identifying covalent targets of antitumor drugs according to claim 3, characterized in that, The breast cancer cells were MDA-MB-231 cells.

5. The method for identifying covalent targets of antitumor drugs according to claim 4, characterized in that, The concentration of the 4-iodo-3-nitrobenzamide is 30 μM; The cell lysis buffer consisted of 50 mM HEPES, 0.1% nuclease, 1× protease inhibitor, 2% sodium dodecyl sulfate, and 8 M urea. The DBIA lysis buffer consisted of 50 mM HEPES, 2% sodium dodecyl sulfate, 8 M urea, and 2 mM DBIA. The final concentration of added dithiothreitol is 8-12 mM, preferably 10 mM; the final concentration of added iodoacetamide is 28-32 mM, preferably 30 mM; and the final concentration of added dithiothreitol is 23-28 mM, preferably 25 mM.

6. The method for identifying covalent targets of antitumor drugs according to claim 1 or 5, characterized in that, The specific steps of enzymatic digestion and affinity enrichment in step S2 are as follows: the labeled protein is digested with enzymes to obtain peptide samples; then streptavidin magnetic beads are used to enrich the peptide samples to obtain enriched peptide samples.

7. The method for identifying covalent targets of antitumor drugs according to claim 1, characterized in that, The specific steps of the quantitative mass spectrometry analysis in step S3 are as follows: the enriched peptide sample is eluted to obtain dried peptides; the dried peptides are then acquired by LC-MS / MS, data is searched and processed, and then differential site screening and spectrum verification are performed to obtain the binding sites of 4-iodo-3-nitrobenzamide. The screening threshold for the differential site selection is Fold change ≥ 4 and p -value < 0.

05.

8. The method for identifying covalent targets of antitumor drugs according to claim 7, characterized in that, The database used for data processing is UniProt-human-filtered-reviewed, the enzyme digestion method is Trypsin / P, and the variable modifications are DBIA-Cys[C], Carbamidomethyl[C], Acetyl[Protein N-term], and Oxidation[M]. The maximum missed cleavage is 2, and the FDR is 1%.

9. The method for identifying covalent targets of antitumor drugs according to claim 1 or 8, characterized in that, The specific steps of the pathway enrichment analysis in step S4 are as follows: the target protein of the binding site is subjected to GO pathway enrichment analysis and KEGG pathway enrichment analysis, and then Fisher's exact test is used to obtain the covalent target of 4-iodo-3-nitrobenzamide. The significance threshold for the Fisher exact test is set to FDR < 0.

05.

10. The use of the method according to any one of claims 1 to 9 for identifying covalent targets of antitumor drugs.

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