A method for screening potential covalent drugs

Abstract

The present invention relates to a method for screening potential covalent drugs, comprising: establishing a qualitative analysis method for covalent adducts formed by a validated drug or a metabolite of a validated drug and acetylcysteine respectively through liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS); incubating a drug to be tested and acetylcysteine in vitro to obtain a first incubation solution; detecting the first incubation solution by the qualitative analysis method; and determining the drug to be tested as a potential covalent drug according to the detection result. The screening method established by the present invention can simply, rapidly and sensitively detect compounds that covalently modify acetylcysteine, providing important candidate compounds for the research and development of covalent drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analytical chemistry, and particularly relates to a method for screening potential covalent drugs. Background Art

[0002] Covalent drugs are drugs containing potentially reactive electrophilic warheads that can bind to target proteins in a covalent bond form. Covalent drugs have advantages such as strong and long-lasting effects, reduced dosage, and reduced dosing frequency. In recent years, with the successive approval and marketing of a variety of targeted covalent inhibitors (TCI) at home and abroad, such as afatinib, ibrutinib, and zanubrutinib, covalent drugs have become the focus of new drug development research.

[0003] Currently, structure-based computer-aided covalent virtual screening has become a method for discovering covalent drugs. However, this method often produces a large number of false positive results and requires further experimental verification. Therefore, it cannot be used as an independent technology, and this method is based on existing small molecule databases, which limits the discovery of structurally diverse and novel covalent drugs.

[0004] Methods for experimentally studying the covalent binding of compounds to target proteins include X-ray crystallography, nuclear magnetic resonance, activity-based protein profiling (ABPP), immunoprecipitation, isotope labeling, etc. However, these methods focus more on studying the protein targets of known covalent compounds or how to interact with proteins, and are not suitable for screening covalent drugs. Moreover, these methods require the synthesis of complex probe compounds with sensitive structures, which is time-consuming and has a long cycle.

[0005] Therefore, how to compress the experimental time while increasing the identification quantity and build a simple, rapid, and sensitive analysis method is the current technical bottleneck that needs to be urgently broken through. Summary of the Invention

[0006] The present invention aims to at least partly solve one of the technical problems existing in the prior art. To this end, the present invention provides a method for screening potential covalent drugs.

[0007] According to one aspect of the present invention, there is provided a method for screening potential covalent drugs, including: establishing a qualitative analysis method for covalent adducts formed by a validation drug or a validation drug metabolite and acetylcysteine respectively through liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS), wherein the validation drug contains a reactive electrophilic warhead; in vitro incubating a drug to be tested and the acetylcysteine to obtain a first incubation solution; detecting the first incubation solution through the qualitative analysis method; and determining the drug to be tested as a potential covalent drug according to the detection result.

[0008] Preferably, a qualitative analysis method for validating covalent adducts formed by a drug or a drug metabolite with N-acetylcysteine is established by liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS), including: determining the drug to be validated that can form the covalent adduct with the N-acetylcysteine; incubating the drug to be validated and the N-acetylcysteine in vitro to obtain a second incubation solution; establishing a qualitative analysis method for the second incubation solution by the liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS).

[0009] Preferably, the establishment of the qualitative analysis method for the second incubation solution by the liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS) includes: calculating the precursor ion information of the covalent adducts formed by the drug to be validated or the drug metabolite with the N-acetylcysteine respectively; introducing the second incubation solution into the ion source of the liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS); fragmenting specific precursor ions according to the precursor ion information to obtain daughter ions; when daughter ion fragments with specific mass-to-charge ratios are detected from the daughter ions, determining the conditions in the qualitative analysis method.

[0010] Preferably, the conditions for fragmenting the specific precursor ions in the qualitative analysis method are: fragmentation voltage: 380 V; collision energy: 35 ± 15 eV; daughter ion resolution: Unit; dwell time: 100 ms.

[0011] Preferably, the daughter ion fragments with the specific mass-to-charge ratios are one or more of m / z 130.0, m / z 162.0, and m / z 164.0.

[0012] Preferably, the chromatographic conditions for the qualitative analysis method are: the chromatographic column is a reversed-phase ultra-high performance liquid chromatography column, mobile phase A is water containing formic acid, mobile phase B is acetonitrile containing formic acid, and gradient elution is performed.

[0013] Preferably, the chromatographic conditions are: the chromatographic column is ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm); mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid; gradient elution is performed in the following manner: 0 - 0.5 min, 5% B; 0.5 - 2 min, 5% - 20% B; 2 - 4 min, 20 - 30% B; 4 - 6 min, 30% - 50% B; 6 - 8 min, 50% - 80% B; 8 - 10 min, 80% - 95% B; 10 - 11.5 min, 95% B; 11.5 - 12 min 95% - 5% B; flow rate is 0.25 mL / min; column temperature is 30°C; injection volume is 1 μL.

[0014] Preferably, the mass spectrometry conditions for the qualitative analysis method are as follows: the ion source is an electrospray ionization source ESI; the MRM scanning mode is positive ion scanning; the drying gas flow rate is 12 - 15 L / min; the nebulizer temperature is 150 - 250 °C; the nebulizer voltage is 20 - 25 psi; the sheath gas flow rate is 9 - 11 L / min; the sheath gas temperature is 200 - 300 °C; the capillary voltage is 3500 - 6000 V, and the nozzle voltage is 0 - 500 V.

[0015] Preferably, the mass spectrometry conditions are as follows: the drying gas flow rate is 13 L / min; the nebulizer temperature is 225 °C; the nebulizer voltage is 25 psi; the sheath gas flow rate is 12 L / min; the sheath gas temperature is 275 °C; the capillary voltage is 4000 V, and the nozzle voltage is 350 V.

[0016] Preferably, after determining that the drug to be tested is a potential covalent drug according to the detection result, it further includes: characterizing the first incubation solution to determine the modification mode of the potential covalent drug to cysteine.

[0017] Preferably, the first incubation solution is characterized by liquid chromatography-high resolution mass spectrometry (UHPLC-Q-TOF-MS / MS), nuclear magnetic resonance (NMR), and high resolution mass spectrometry to determine the modification mode of the potential covalent drug to the cysteine.

[0018] Preferably, the in vitro incubation of the drug to be tested and the acetylcysteine includes: incubating the drug to be tested and the acetylcysteine in a potassium phosphate buffer solution containing NADPH solution and liver microsomes, wherein the concentration of the liver microsomes is 1.0 mg / mL, the concentration of the drug to be tested is 50 μM, the concentration of the acetylcysteine is 5 mM, the total incubation volume is 200 μL, the incubation temperature is 37 °C, and the incubation time is 2 hours.

[0019] Preferably, after the in vitro incubation of the drug to be tested and the acetylcysteine, it further includes: vortex-mixing the first incubation solution; centrifuging the first incubation solution at 4 °C and 15000 g for 10 min to obtain a first supernatant; drying the first supernatant under a nitrogen atmosphere to obtain a precipitate; redissolving the precipitate in 50% methanol and centrifuging again to obtain a second supernatant for detection.

[0020] Preferably, the verification drugs include triptolide, curcumin, osimertinib, toosendanin, naringenin.

[0021] Preferably, the drugs to be tested include celastrol, bufotalin, ibrutinib, sunitinib, ciprofloxacin, gatifloxacin, trovafloxacin, ozagrel, ketamine.

[0022] Preferably, the reactive electrophilic warhead is one or more of an oxirane ring, an α,β-unsaturated carbonyl group, a furan ring, a benzene ring, a quinone precursor, an alkaloid, cyclohexanone, hydroxycyclohexane, thiophene, or an aromatic amine.

[0023] Preferably, the in vitro incubation of the drug with acetylcysteine refers to a reaction system that generates metabolism of the substrate, such as microsomes, S9, hepatocytes, recombinant metabolic enzymes, etc. Acetylcysteine is used as a capture reagent to capture the drug and its metabolites, forming a stable adduct for easy mass spectrometry detection.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The potential covalent drug screening method established by the present invention can simply, quickly, and sensitively detect compounds that covalently modify cysteine, providing important candidate compounds for the research and development of covalent drugs. In addition, this method is applicable to the detection of compound-acetylcysteine adducts of various structural types, has a wide range of substrate applicability, and provides new ideas and methods for the design of covalent drugs. This method can also play an important role in the study of drug action mechanisms and can apply the identified covalent modification methods to the identification of modified proteins. Description of the Drawings

[0025] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 shows the chemical structural formula of a validated drug containing a reactive electrophilic warhead;

[0027] Figure 2 shows the chemical structural formula of the drug to be tested;

[0028] Figure 3 shows the MRM chromatogram of a covalent adduct formed by a part of the drug or its metabolite and acetylcysteine; and

[0029] Figure 4 shows the MRM chromatogram of a covalent adduct formed by another part of the drug or its metabolite and acetylcysteine. Detailed Description of the Invention

[0030] The following listed examples are to enable those skilled in the art to better understand the present invention. It should be noted that the following examples do not limit the scope of protection required by the present invention and are only illustrative examples. The raw materials, reagents, or devices mentioned in the following examples can be obtained from commercial sources or by known existing methods without special instructions.

[0031] For the development of new covalent drugs, the covalent-binding target proteins are currently identified mostly at the proteome level. Cysteine has received great attention due to its inherent high nucleophilicity. Most targeted covalent inhibitors can react with the sulfhydryl group on the cysteine residue of proteins through reactive electrophilic groups to form covalent bonds, thereby changing the protein structure and function. In addition, cysteine in proteins is the functional active site of many enzymes and is involved in many physiological processes, such as kinases, tyrosine phosphatases, proteases, deubiquitinases, oxidoreductases, and acyltransferases. Based on these characteristics, cysteine residues on proteins have become the focus of new drug research and development and are the research object of the present invention. Specifically, the present invention uses acetylcysteine to simulate cysteine residues on proteins as a capture reagent to obtain more candidate covalent compounds.

[0032] Example 1

[0033] According to an embodiment of the present invention, a method for screening potential covalent drugs is provided, including: establishing a qualitative analysis method for validating covalent adducts formed by a validation drug or a metabolite of the validation drug and acetylcysteine respectively through liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS), wherein the validation drug contains a reactive electrophilic warhead; incubating the drug to be tested and acetylcysteine in vitro to obtain a first incubation solution; detecting the first incubation solution through the qualitative analysis method; and determining the drug to be tested as a potential covalent drug according to the detection result.

[0034] The so-called validation drug refers to a drug that has been reported in the existing literature to be able to react with cysteine to form a covalent bond, and this drug has the same or similar physicochemical properties as covalent drugs.

[0035] The so-called reactive electrophilic warhead refers to an electrophilic group existing in a compound that undergoes a covalent reaction with the nucleophilic cysteine on a protein. This electrophilic group is the basis for covalently modifying proteins, and the electrophilic warhead can be one or more of an oxirane ring, an α,β-unsaturated carbonyl, a furan ring, a benzene ring, a quinone precursor, an alkaloid, a cyclohexanone, a hydroxycyclohexane, a thiophene, or an aromatic amine. Figure 1 The chemical structural formula of the validation drug containing an electrophilic warhead determined in the present invention is specifically shown.

[0036] Specifically, first, a drug that can form a covalent adduct with cysteine is determined as a validation drug; then, the validation drug and acetylcysteine are incubated in vitro to obtain a second incubation solution; and a qualitative analysis method for the second incubation solution is established through liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS).

[0037] The so-called in vitro incubation to verify the drug and acetylcysteine and obtain the second incubation solution refers to incubating the mixture of the drug and the metabolite formed by the drug and acetylcysteine and the covalent adduct of cysteine through the in vitro metabolic enzyme system. The specific steps are as follows: The verified drug (50 μM) and acetylcysteine (5 mM) are incubated in a potassium phosphate buffer solution containing NADPH solution and liver microsomes (1.0 mg / mL) at 37 °C for 2 hours. The total incubation volume is 200 μL. After vortex mixing, it is centrifuged at 4 °C and 15,000 rpm for 10 min. The supernatant is collected, dried under nitrogen, redissolved in 50% methanol, and the supernatant is taken after centrifugation for LC-QQQ-MS analysis.

[0038] More specifically, the method for establishing the qualitative analysis of the second incubation solution by liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS) specifically includes: First, calculate the precursor ion information of the covalent adducts formed by the verified drug or the metabolite of the verified drug and acetylcysteine respectively; Second, introduce the second incubation solution into the ion source of liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS); Then, fragment specific precursor ions according to the precursor ion information to obtain daughter ions; When daughter ion fragments with specific mass-to-charge ratios are detected from the daughter ions, determine the conditions in the qualitative analysis method.

[0039] The present invention provides the specific steps and experimental data for establishing the qualitative analysis method.

[0040] (1) Establish a database of covalent adducts formed by the verified drug itself or the metabolite formed after phase I / II biotransformation with acetylcysteine. This database contains relevant information of the precursor ions of the covalent adducts, such as the precursor mass-to-charge ratio. The details of this database are shown in Table 1.

[0041] Table 1 Database of covalent adducts of the verified drug-acetylcysteine

[0042]

[0043] (2) Introduce the second incubation solution into the ion source of liquid chromatography-triple quadrupole-mass spectrometry (LC-QQQ-MS), fragment the precursor ions included in the database to obtain daughter ions, and adopt the MRM scanning mode to remove the interference of other ions.

[0044] (3) When specific daughter ion fragments are detected from the daughter ions, determine the conditions in the qualitative analysis method. This process is essentially to continuously optimize the detection conditions and detect the daughter ions of m / z 130.0, m / z 162.0, and m / z 164.0. The identification of covalent adducts is divided into three levels, all of which are recognized as verifying the drug-N-acetylcysteine covalent adduct: (1) Detect all three daughter ions of m / z 130.0, m / z 162.0, and m / z 164.0 simultaneously; (2) Detect any two of the three daughter ions of m / z 130.0, m / z 162.0, and m / z 164.0 simultaneously; (3) Detect any one of the three daughter ions of m / z 130.0, m / z 162.0, and m / z 164.0; the signal-to-noise ratio is greater than 3.

[0045] The chromatographic conditions are set as follows: Use a 6490-Q-TOF-MS mass spectrometer, a Waters ACQUITY BEH C18 column (2.1×100 mm, 1.7 μm) for identifying the drug-cysteine adduct. The flow rate is 0.25 mL / min, the mobile phase A is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid in acetonitrile. Gradient elution: 0 - 0.5 min, 5% B; 0.5 - 2 min, 5% - 20% B; 2 - 4 min, 20 - 30% B; 4 - 6 min, 30% - 50% B; 6 - 8 min, 50% - 80% B; 8 - 10 min, 80% - 95% B; 10 - 11.5 min, 95% B; 11.5 - 12 min 95% - 5% B; the injection volume is 1 μL.

[0046] The mass spectrometry conditions are set as follows: Ion source: electrospray ionization source ESI; Scanning mode: MRM positive ion scanning mode; Dry gas flow rate 13 L / min; Nebulizer temperature 225 °C; Nebulizer voltage 25 psi; Sheath gas flow rate 12 L / min; Sheath gas temperature 275 °C; Capillary voltage 4000 V, nozzle 350 V; MRM parameter settings: Precursor ion acquisition is based on the established database; Daughter ions are set as the characteristic ion fragments of N-acetylcysteine, m / z130.0, m / z 162.0, and m / z 164.0; Daughter ion resolution: Unit; Dwell time: 100 ms; Fragmentation voltage: 380 V; Collision energy: 30 ± 15 eV.

[0047] The mass spectrometry data of the covalent adducts formed by triptolide, curcumin, osimertinib, toosendanin, naringenin, etc. with N-acetylcysteine are shown in Table 2, indicating that this method can achieve rapid screening of covalent compounds by using the precursor information of cysteine adducts and the information of characteristic daughter ion fragments.

[0048] Table 2 Validation of MRM Mass Spectrometry Data of Covalent Adducts Formed by Drugs or Their Metabolites and Acetylcysteine

[0049]

[0050]

[0051] Example 2

[0052] This example presents the specific application of using the optimized qualitative analysis method to rapidly screen for potential covalent drugs.

[0053] First, incubate the drug to be tested and acetylcysteine in vitro to obtain the first incubation solution; second, detect the first incubation solution by the optimized qualitative analysis method. When the test result shows characteristic values, it can be determined that the drug to be tested or its metabolite is a potential covalent drug.

[0054] (1) Based on the strategy of drug repurposing, screen potential cysteine - targeting drugs from existing compounds as candidates. This method can be applied to the detection of covalent adducts formed by celastrol, bufotalin, ibrutinib, sunitinib, ciprofloxacin, gatifloxacin, trovafloxacin, ozagrel, ketamine and other drugs to be tested with acetylcysteine. Figure 2 The chemical structural formula of the drug to be tested is shown.

[0055] (2) Based on metabolic and biotransformation pathways (metabolites or metabolic reaction intermediates formed after phase I / II biotransformation), establish a database of possible covalent conjugates formed by the drug to be tested and acetylcysteine, which contains information such as the precursor ions of the covalent adduct.

[0056] (3) Incubate the drug to be tested and acetylcysteine in vitro to obtain the first incubation solution, aiming to prepare covalent adducts formed by the drug to be tested or the metabolites formed after phase I / II biotransformation of the drug to be tested and acetylcysteine respectively. The specific steps are as follows: The drug (50 μM) and acetylcysteine (5 mM) are incubated in a potassium phosphate buffer solution containing NADPH solution and liver microsomes (1.0 mg / mL) at 37 °C for 2 hours. The total incubation volume is 200 μL. After vortex mixing, centrifuge at 4 °C, 15000 rpm for 10 min, collect the supernatant, dry it under nitrogen, redissolve it in 50% methanol, centrifuge and take the supernatant for LC - QQQ - MS analysis.

[0057] (4) Chromatography - Mass Spectrometry Conditions:

[0058] The chromatographic conditions were as follows: A 6490-Q-TOF-MS mass spectrometer was used, and the chromatographic column was Waters ACQUITY BEH C18 column (2.1×100 mm, 1.7 μm) to identify the drug and acetylcysteine adducts. The flow rate was 0.25 mL / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. Gradient elution: 0 - 0.5 min, 5% B; 0.5 - 2 min, 5% - 20% B; 2 - 4 min, 20 - 30% B; 4 - 6 min, 30% - 50% B; 6 - 8 min, 50% - 80% B; 8 - 10 min, 80% - 95% B; 10 - 11.5 min, 95% B; 11.5 - 12 min, 95% - 5% B; the injection volume was 1 μL.

[0059] The mass spectrometry conditions were as follows: Ion source: electrospray ionization source ESI; Scanning mode: MRM positive ion scanning mode; Dry gas flow rate 13 L / min; Nebulizer temperature 225 °C; Nebulizer voltage 25 psi; Sheath gas flow rate 12 L / min; Sheath gas temperature 275 °C; Capillary voltage 4000 V, nozzle 350 V; MRM parameter settings: Precursor ion acquisition was based on the established database; Product ions were set as the characteristic ion fragments of acetylcysteine m / z 130.0, m / z 162.0, and m / z 164.0; Product ion resolution: Unit; Dwell time: 100 ms; Fragmentation voltage: 380 V; Collision energy: 30 ± 15 eV.

[0060] (5) The identification of the covalent adducts formed by the drug to be tested or the metabolites formed after phase I / II biotransformation of the drug to be tested with acetylcysteine was carried out at three levels. (1) The covalent adducts were confirmed by the three product ions m / z 130.0, m / z 162.0, and m / z 164.0 in the MRM method; (2) The covalent adducts were confirmed by two product ions in the MRM method; (3) The covalent adducts were confirmed by one product ion in the MRM method; The signal-to-noise ratio was greater than 3, and all of the above were identified as the covalent adducts of the drug to be tested or its metabolites with acetylcysteine.

[0061] The results showed that potential drugs to be tested or their metabolites formed covalent adducts with acetylcysteine, and the detailed mass spectrometry data are shown in Table 3.

[0062] Table 3 MRM mass spectrometry data of the covalent adducts formed by the drug to be tested or its metabolites with acetylcysteine

[0063]

[0064]

[0065] (6) A rapid screening strategy for potential covalent drugs based on LC-QQQ-MS technology successfully detected covalent conjugates in reaction systems of various types of compounds with N-acetylcysteine, and had high response signals. Using this method, the interference of the matrix to the target substance can be effectively reduced, the detection sensitivity can be improved, and it is beneficial to the screening of trace covalent adducts in samples.

[0066] Example 3

[0067] Characterize and verify the covalent adducts formed by the potential covalent drugs or their metabolites and N-acetylcysteine screened out, and confirm the structures of the potential covalent drugs.

[0068] (1) Preparation of the covalent adduct of the drug-N-acetylcysteine: The drug (50 μM) and N-acetylcysteine (5 mM) were incubated in a potassium phosphate buffer solution containing NADPH solution and liver microsomes (1.0 mg / mL) at 37 °C for 2 hours. The total incubation volume was 200 μL. After vortex mixing, centrifuge at 4 °C, 15000 for 10 min, collect the supernatant, dry it under nitrogen, redissolve it in 50% methanol, centrifuge and take the supernatant for UHPLC-Q-TOF-MS / MS analysis.

[0069] (2) UHPLC-Q-TOF-MS / MS chromatographic-mass spectrometric conditions:

[0070] Chromatographic conditions: Use a 6545-Q-TOF-MS mass spectrometer, Waters ACQUITY BEH C18 column (2.1×100 mm, 1.7 μm) to identify the reaction adducts. The flow rate was 0.25 mL / min, mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; gradient elution: 0 - 0.5 min, 5% B; 0.5 - 2 min, 5% - 20% B; 2 - 4 min, 20 - 30% B; 4 - 6 min, 30% - 50% B; 6 - 8 min, 50% - 80% B; 8 - 10 min, 80% - 95% B; 10 - 11.5 min, 95% B; 11.5 - 12 min 95% - 5% B; the flow rate was 0.25 mL / min; the column temperature was 30 °C; the injection volume was 1 μL.

[0071] Mass spectrometric conditions: The ion source was an electrospray ionization source ESI; the drying gas flow rate was 9 L / min; the nebulizer temperature was 325 °C; the nebulizer voltage was 35 psi; the sheath gas temperature was 350 °C; the sheath gas flow rate was 11 L / min; the nozzle voltage was 3500 V, and the collision energy was 15 - 40 eV.

[0072] (3) Identify the covalent adduct of the drug-N-acetylcysteine screened out above by tandem mass spectrometry, and the main characteristic secondary fragment ions are shown in Table 4.

[0073] High-resolution mass spectrometry data of covalent adducts formed by drugs or their metabolites and N-acetylcysteine in Table 4

[0074]

[0075]

[0076]

[0077] (4) The above results indicate that the structure and modification mode of the covalent adducts were confirmed by high-resolution mass spectrometry analysis of the covalent adducts of drugs or their metabolites - N-acetylcysteine screened by the LC-QQQ-MS method, further verifying that the method of rapidly screening potential covalent drugs by combining LC-QQQ-MS technology with a database is reliable and effective.

[0078] Example 4

[0079] Confirmation of potential covalent drugs at the protein level.

[0080] (1) Sample preparation:

[0081] After incubating the drug (50 μM) with the target protein in a liver microsome (1.0 mg / mL) incubation system at 37 °C for 2 hours, centrifuge at 4 °C and 10,000 g for 5 minutes, and collect the supernatant; add 4 volumes of acetone to the supernatant, centrifuge at 4 °C and 10,000 g for 5 minutes, and collect the precipitate; dissolve the precipitate in 8 M urea, centrifuge to remove insoluble matter, take 100 μg of protein sample and dilute it to 100 μL with MilliQ water, add 5 μL of 200 mM DTT, and incubate at room temperature for 1 hour. Add 4 μL of 1 M IAA, and incubate at room temperature in the dark for 1 hour. Add 20 μL of 200 mM DTT, incubate at room temperature for 1 hour to terminate the reaction. Add 2 μg of trypsin, and incubate at 37 °C overnight. Desalt the enzymatically digested sample with SPE C18. Collect the eluate, finally dry the eluate with nitrogen, redissolve it in 50 μL of 2% acetonitrile containing 0.1% trifluoroacetic acid, centrifuge, and analyze by 2D-nano-LC-Q-TOF-MS method.

[0082] (2) 2D-nano-LC-Q-TOF-MS method:

[0083] Chromatographic conditions: The enzymatically digested polypeptides were first enriched on a strong cation exchange chromatography column, Poros 10S nanoViper SCX (300 μm × 100 mm, 10 μm), and then eluted with ammonium acetate at concentrations of 5, 10, 25, 50, and 100 mM and a pH of approximately 2.7. Each eluate had a volume of 20 μL. Then the eluate was transferred to an Acclaim PepMap RSLC nanoViper C18 column (75 μm × 150 mm, 2 μm). The chromatographic conditions were as follows: For the A phase of the Loading pump, it was a 2% acetonitrile solution containing 0.05% trifluoroacetic acid, with a flow rate of 5 μL / min; for the A phase of the NC pump, it was an aqueous solution containing 0.1% formic acid, and the B phase was an acetonitrile solution containing 0.1% formic acid, with a flow rate of 0.3 μL / min. The gradient change of the NC pump was as follows: 0–8 min, 5% B; 8–110 min, increase the B phase to 30%; 110–110.1 min, increase B to 80% and maintain it until 115 min; then reduce the proportion of the B phase to 5% within 0.1 min and maintain it for 5 min; the injection volume was 1 μL.

[0084] Mass spectrometry conditions: The mass spectrometry was performed using a Bruker MaXis impact Q-TOF mass spectrometer, with a Captive spray ion source, in positive ion mode. The parameter settings were as follows: the end plate offset was 500 V, the capillary voltage was 1600 V, the drying gas flow rate and temperature were 4.0 L / min and 160 °C respectively, the quadrupole ion energy was 5 eV, the CE was 8 eV, and the top 10 precursor ions were selected within the range of m / z 300 to 1700 per second, excluding single-charged ions, and precursor ions with 2 + ,3 + ,4 + or 3 or 4 charges were preferentially introduced into MS / MS analysis.

[0085] (3) Identification of modified proteins:

[0086] The modification methods and sites of the drugs and their active metabolites identified in Examples 2 and 3 were added to the proteomics database as variable modifications for database searching to identify the modified proteins. See Table 5 for details.

[0087] Table 5 Proteins modified by triptolide and its metabolites

[0088]

[0089] The present invention utilizes an ultra-high performance liquid chromatography system and a mass spectrometer, combines a database and MRM technology, selectively performs collision-induced dissociation on the parent ions of covalent drugs to remove the interference of other ions, and specifically screens out the characteristic daughter ions of covalent drugs, thereby achieving sensitive, specific, and accurate screening of drug-N-acetylcysteine adducts.

[0090] In summary, the LC-QQQ-MS method established by the present invention can simply, rapidly, and sensitively detect compounds that covalently modify cysteine, providing important candidate compounds for the research and development of covalent drugs. In addition, this method is applicable to the detection of compound-N-acetylcysteine adducts of various structural types, has a wide substrate applicability, provides new ideas and methods for the design of covalent drugs. In addition, it can also play an important role in the study of drug action mechanisms. The identified covalent modification methods can also be applied to the identification of modified proteins.

[0091] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for screening potential covalent drugs, characterized in that Comprising: By liquid chromatography - triple quadrupole - mass spectrometry (LC - QQQ - MS), a qualitative analysis method for validating covalent adducts formed between a drug or a drug metabolite and N - acetylcysteine is established, including: Identifying the validation drug containing a reactive electrophilic warhead; In vitro incubating the validation drug and the N - acetylcysteine to obtain a second incubation solution; By the liquid chromatography - triple quadrupole - mass spectrometry (LC - QQQ - MS), a qualitative analysis method for the second incubation solution is established, including: Calculating the precursor ion information of the covalent adducts formed between the validation drug or the validation drug metabolite and the N - acetylcysteine respectively; Introducing the second incubation solution into the ion source of the liquid chromatography - triple quadrupole - mass spectrometry (LC - QQQ - MS), and the scanning mode of the mass spectrometry conditions is MRM positive ion scanning; Fragmenting specific precursor ions according to the precursor ion information to obtain daughter ions; When a fragment of a daughter ion with a specific mass-to-charge ratio is detected from the daughter ions, various conditions in the qualitative analysis method are determined, where the fragment of the daughter ion with the specific mass-to-charge ratio is m / z 130.0, m / z 162.0 and m / z one or more of 164.0; In vitro incubating the drug to be tested and the N - acetylcysteine to obtain a first incubation solution; Detecting the first incubation solution by the qualitative analysis method; Determining that the drug to be tested is a potential covalent drug according to the detection result.

2. The method according to claim 1, wherein The conditions for fragmenting the specific precursor ions in the qualitative analysis method are: Fragmentation voltage: 380 V; Collision energy: 35 ± 15 eV; Daughter ion resolution: Unit; Dwell time: 100 ms.

3. The method according to claim 1, characterized in that, The chromatographic conditions of the qualitative analysis method are: The chromatographic column is a reversed - phase ultra - high performance liquid chromatography column. Mobile phase A is water containing formic acid, and mobile phase B is acetonitrile containing formic acid, and gradient elution is performed.

4. The method according to claim 3, wherein The chromatographic conditions are: The chromatographic column is ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 µm; Mobile phase A is water containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid; Gradient elution is performed as follows: 0 - 0.5 min, 5% B; 0.5 - 2 min, 5% - 20% B; 2 - 4 min, 20 - 30% B; 4 - 6 min, 30% - 50% B; 6 - 8 min, 50% - 80% B; 8 - 10 min, 80% - 95% B; 10 - 11.5 min, 95% B; 11.5 - 12 min, 95% - 5% B; flow rate is 0.25 mL / min; column temperature is 30°C; injection volume is 1 μL.

5. The method according to claim 1, characterized in that, The mass spectrometry conditions of the qualitative analysis method are: The ion source is an electrospray ionization source (ESI); the dry gas flow rate is 12 - 15 L / min; the nebulizer temperature is 150 - 250°C; the nebulizer voltage is 20 - 25 psi; the sheath gas flow rate is 9 - 11 L / min; the sheath gas temperature is 200 - 300°C; the capillary voltage is 3500 - 6000 V, and the nozzle voltage is 0 - 500 V.

6. The method according to claim 5, characterized in that, The mass spectrometry conditions are: The drying gas flow rate is 13 L / min; the atomizer temperature is 225 °C; the atomizer voltage is 25 psi; the sheath gas flow rate is 12 L / min; the sheath gas temperature is 275 °C; the capillary voltage is 4000 V, and the nozzle voltage is 350 V.

7. The method according to any one of claims 1 to 6, characterized in that, After determining that the drug to be tested is a potential covalent drug according to the detection results, it further includes: Characterize the first incubation solution to determine the modification mode of the potential covalent drug to cysteine.

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