Alkannin action target screened based on LIP-MS technology and application

Abstract

The invention discloses an alkannin acting target spot screened based on an LIP-MS technology and application, and relates to the technical field of medicines.The alkannin acting target spot is technically characterized in that a protein sample is subjected to restrictive enzymolysis through wide specific protease, and the characteristic that the conformation stability of a binding region peptide fragment can be improved through binding of a medicine and target protein is utilized; further, trypsin is used for carrying out secondary enzymolysis, a quantitative proteomics technology is used for collecting peptide fingerprint spectrums, specific conformation maintaining peptide fragments obtained after drug treatment are screened out by analyzing and comparing changes of the peptide fingerprint spectrums of a control group and a drug treatment group, target protein information corresponding to the peptide fragments is identified through database matching, and compared with a traditional DARTS method, the method has the advantages that the method is simple and convenient, and the efficiency is high. According to non-labeled drug target analysis based on peptide fingerprint spectrum, a detected object is converted into a peptide fragment from a protein, and the interaction between a low-affinity multi-target drug and the protein, low-abundance protein and local conformation change information of high-molecular-weight protein are expected to be represented, so that the accuracy and sensitivity of target analysis are improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a shikonin action target screened based on LIP-MS technology and an application thereof. Background Art

[0002] Shikonin (SK) is a fat-soluble naphthoquinone compound extracted from the traditional Chinese medicine Lithospermum officinale. It has multiple pharmacological activities such as anti-inflammatory, antioxidant, antiviral, and antifungal. Studies have found that shikonin has strong anti-Candida albicans activity, especially the antifungal effect on fluconazole-resistant bacteria. The combination of shikonin with azoles and polyene drugs can reduce the dosage of these drugs and restore their antibacterial effects on drug-resistant fungi. It is a good antifungal drug synergist. However, which targets in Candida albicans cells can shikonin directly act on, and by what mechanism can it regulate upstream and downstream genes or pathways to exert its antifungal effect? ​​This question has not been well answered.

[0003] Drug targets are the key biological basis for drug activity. The discovery and identification of drug targets has always been a research hotspot in various fields of pharmacy. In recent years, a variety of labeled and non-labeled drug target analysis methods have been developed. Relatively speaking, non-labeled drug target identification does not require the structure-activity relationship and label modification of small molecules, which greatly shortens the time of target analysis. Therefore, it has been deeply studied and widely used. Non-labeled drug target identification methods mainly include DARTS (drug affinity response target stability analysis), SPROX (protein stability assessment based on oxidation rate) and CETSA (cell thermal shift analysis). These methods identify drug targets by observing the changes in biophysical properties (such as stability, oxidation rate or thermal stability) caused by the binding of drugs to target proteins. However, these methods also have certain limitations. First, DARTS technology has limited ability to detect low-abundance targets, and the hydrolysis sensitivity of target proteins and the specificity of selected proteases may affect the results. Secondly, SPROX technology is limited by the content of methionine in proteins, and is currently mainly suitable for the analysis of cell lysates and cannot be directly applied to living cell systems. In addition, CETSA technology requires higher drug concentrations, is time-consuming and costly, and lacks information on the binding of the drug to the specific structural domain of the target protein.

[0004] To this end, the present invention aims to provide a target of shikonin and its application screened based on LIP-MS technology to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems, provide a target of shikonin based on LIP-MS technology screening and application, adopt a non-label drug target analysis method based on peptide fingerprint spectrum, extract the total protein of Candida albicans cells under the intervention of shikonin, use Proteinase K to perform restriction enzymatic hydrolysis, compare the changes of peptide fingerprint spectrum of control group and drug treatment group, screen out the exclusive "conformation-maintaining peptide segment" in the shikonin treatment group, and identify the target protein of shikonin inhibiting Candida albicans biofilm through database matching.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a target of shikonin based on LIP-MS technology screening, and the technology comprises the following steps:

[0008] S1. Protein extraction: monoclonal Candida albicans was inoculated into 1 mL YPD liquid culture medium and cultured at 30°C and 200 rpm for 16 hours; after the culture, the cells were washed three times with PBS, a total of 5 mL of protein extraction buffer was added, and mixed with twice the volume of acidified glass beads, and the protein was extracted in an ice bath, and the protein concentration was detected using a BCA protein concentration determination kit, and diluted to 1.0 mg / ml with PBS, and stored in a -80°C refrigerator for use;

[0009] S2, limited proteolysis: take 100 μg of diluted protein solution and add appropriate concentration of drug or DMSO, add proteinase K to the sample at an enzyme / substrate ratio of 1:100, incubate the sample for 3 minutes, and then heat to inactivate PK; then add sodium deoxycholate solution to a final concentration of 5%, dithiothreitol to a final concentration of 12 mM, and iodoacetamide to a final concentration of 40 mM to alkylate the reduced cysteine ​​residues; add trypsin at an enzyme / substrate ratio of 1:100 wt / wt, and incubate at 37°C overnight; stop digestion and precipitate sodium deoxycholate by adding 98% vol / vol formic acid to a final concentration of 2% vol / vol; finally, high speed for 10 minutes to remove the precipitate, and after desalting, transfer the supernatant for mass spectrometry analysis;

[0010] S3. Mass spectrometry analysis: Mass spectrometry analysis was performed using an ultra-high performance liquid chromatography-mass spectrometry system (nano-UPLC-MS, model: Ekspert nano LC 400 and Triple-TOF 5600+, AB Sciex, USA), which separated peptides on a ChromXP C18 column (0.3 mm × 150 mm, 3 μm, AB Sciex, USA) at a flow rate of 5 μl / min. The peptides were eluted using a 90-min acetonitrile-water gradient. Quantification of the Candida albicans proteome was achieved by SWATH-based mass spectrometry; data were collected and analyzed in 1.7 software and ProteinPilot 4.5; Markerview software and Uniprot database were used to establish a differential protein information table through the Uniprot database; by comparing the differences in effective peptides, the potential targets of shikonin acting on Candida albicans were screened;

[0011] S4. Target verification: Screen and sort the selected targets based on whether they are drug concentration-dependent; through comprehensive analysis of drug structure and target function, confirm Fructose-bisphosphate aldolase as the target, and take corresponding verification measures.

[0012] Provided is an application of a shikonin action target screened based on LIP-MS technology, wherein the Fructose-bisphosphate aldolase target directly acts on shikonin to resist fungi.

[0013] Beneficial effects of this program:

[0014] The present invention proposes to adopt a non-labeling drug target analysis method based on peptide fingerprint spectrum, extract the total protein of Candida albicans cells under the intervention of lithospermum, perform restriction enzymatic hydrolysis with Proteinase K, compare the changes in peptide fingerprint spectrum of the control group and the drug treatment group, screen out the exclusive "conformation-preserving peptide segment" in the shikonin treatment group, and identify the target protein of shikonin that inhibits Candida albicans biofilm through database matching; compared with the traditional DARTS method, the non-labeling drug target analysis based on peptide fingerprint spectrum converts the detection object from protein to peptide segment, which is expected to characterize the low-affinity multi-target drug-protein interaction, low-abundance protein, and local conformational change information of large molecular weight protein, so as to improve the accuracy and sensitivity of target analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a volcano diagram of the SK target protein screening results in the embodiment of the present invention;

[0016] Figure 2is a graph showing the affinity determination between SK and the potential target Fba1 in an embodiment of the present invention;

[0017] Figure 3 The activity of SK in the present invention to inhibit Fba1 (SK concentration 0-32 μg / ml)

[0018] Figure 4 This is a diagram showing the differences between wild bacteria and Fba1 single-arm knockout bacteria under the action of SK in the example of the present invention. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0020] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0021] Example:

[0022] The solution provided by the embodiment of the present invention is as described in the above invention content, and provides a target of shikonin screened based on LIP-MS technology, and the technology comprises the following steps:

[0023] S1. Protein extraction: monoclonal Candida albicans was inoculated into 1 mL YPD liquid culture medium and cultured at 30°C and 200 rpm for 16 hours; after the culture, the cells were washed three times with PBS, a total of 5 mL of protein extraction buffer was added, and mixed with twice the volume of acidified glass beads, and the protein was extracted in an ice bath, and the protein concentration was detected using a BCA protein concentration determination kit, and diluted to 1.0 mg / ml with PBS, and stored in a -80°C refrigerator for use;

[0024] S2, limited proteolysis: take 100 μg of diluted protein solution and add appropriate concentration of drug or DMSO, add proteinase K to the sample at an enzyme / substrate ratio of 1:100, incubate the sample for 3 minutes, and then heat to inactivate PK; then add sodium deoxycholate solution to a final concentration of 5%, dithiothreitol to a final concentration of 12 mM, and iodoacetamide to a final concentration of 40 mM to alkylate the reduced cysteine ​​residues; add trypsin at an enzyme / substrate ratio (wt / wt) of 1:100, and incubate at 37°C overnight; stop digestion and precipitate sodium deoxycholate by adding 98% (vol / vol) formic acid to a final concentration of 2% (vol / vol); finally, high speed for 10 minutes to remove the precipitate, and after desalting, transfer the supernatant for mass spectrometry analysis;

[0025] S3. Mass spectrometry analysis: Mass spectrometry analysis was performed using an ultra-high performance liquid chromatography-mass spectrometry system (nano-UPLC-MS, model: Ekspert nano LC 400 and Triple-TOF 5600+, AB Sciex, USA), which separated peptides on a ChromXP C18 column (0.3 mm × 150 mm, 3 μm, AB Sciex, USA) at a flow rate of 5 μl / min. The peptides were eluted using a 90-min acetonitrile-water gradient. Quantification of the Candida albicans proteome was achieved by SWATH-based mass spectrometry; data were collected and analyzed in 1.7 software and Protein Pilot 4.5; Markerview software and Uniprot database were used to establish a differential protein information table through the Uniprot database; by comparing the differences in effective peptides, potential targets of shikonin on Candida albicans were screened, such as Figure 1 As shown;

[0026] S4. Target verification: The potential targets screened were further screened and ranked according to whether they were drug concentration-dependent, as shown in Table 1. The top three were Fructose-bisphosphate aldolase (Fba1), ATP-dependent RNA helicase eIF4A (TIF1), and Glucose-6-phosphate isomerase (PGI1). Comprehensive analysis of drug structure and target function showed that Fructose-bisphosphate aldolase (Fba1) was the most likely target for SK to inhibit Candida albicans, so corresponding verification was carried out subsequently.

[0027] Table 1 List of target screening based on drug concentration dependence

[0028]

[0029] Verification analysis:

[0030] SK has specific binding activity to Fba1:

[0031] Surface plasmon resonance (SPR) analysis was used to quantitatively evaluate the binding affinity of SK to Fba1. The affinity constant (KD) between SK and Fba1 was determined using a Biacore T200 instrument. The results are shown in Figure 2 The vertical line indicates the -7 Binding affinity of MKD.

[0032] SK inhibits Fba1 activity:

[0033] To test whether the interaction between SK and Fba1 affects the function of Fba1, the inhibitory effect of SK on fructose bisphosphate aldolase (Fba1) activity was performed, and the results are shown in Figure 3 As shown. The half-maximal inhibitory concentration (IC50) of SK in inhibiting Fba1 activity was 4.639±0.253μg / ml.

[0034] The inhibitory effect of SK on mycelium:

[0035] In order to investigate whether the inhibitory effect of SK on mycelial growth is related to Fba1, the differences between wild-type fungi and Fba1 single-arm knockout fungi under the action of SK were compared. Figure 4 As shown, the results showed that compared with the wild-type strain, the Fba1 single-arm knockout bacteria showed more significant growth inhibition under the action of SK. This finding suggests that the presence or activity of Fba1 is essential for alleviating the inhibitory effect of SK. Specifically, Fba1 may act as a direct or indirect target of SK and participate in regulating metabolic pathways or signal transduction networks related to hyphal growth.

[0036] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A target of shikonin screened based on LIP-MS technology, characterized by: The technique comprises the following steps: S1. Protein extraction: monoclonal Candida albicans was inoculated into 1 mL YPD liquid culture medium and cultured at 30°C and 200 rpm for 16 hours; after the culture, the cells were washed three times with PBS, a total of 5 mL of protein extraction buffer was added, and the mixture was mixed with acidified glass beads twice the volume of the cells, and the protein was extracted in an ice bath, and the protein concentration was detected using a BCA protein concentration assay kit, and the mixture was diluted to 1.0 mg / ml with PBS and stored in a -80°C refrigerator for later use; S2. Limited proteolysis: 100 μg of diluted protein solution was added with appropriate concentration of drug or DMSO, and proteinase K was added to the sample at an enzyme / substrate ratio of 1:100, and the sample was incubated for 3 minutes, and then the PK was inactivated by heating; then sodium deoxycholate solution was added to a final concentration of 5%, dithiothreitol was added to a final concentration of 12 mM, and iodoacetamide was added to a final concentration of 40 mM to alkylate the reduced cysteine ​​residues; trypsin was added at an enzyme / substrate ratio of 1:100 wt / wt, and incubated at 37°C overnight; digestion was stopped and sodium deoxycholate was precipitated by adding 98% vol / vol formic acid to a final concentration of 2% vol / vol; finally, the precipitate was removed by centrifugation at 16,000 g for 10 minutes, and after desalting, the supernatant was transferred for mass spectrometry analysis; S3. Mass spectrometry analysis: Quantification of the Candida albicans proteome was achieved by SWATH-based mass spectrometry; data were collected and analyzed in 1.7 software and Protein Pilot 4.5; Markerview software and Uniprot database were used to establish a differential protein information table through the Uniprot database; by comparing the differences in effective peptides, the potential targets of shikonin on Candida albicans were screened; S4. Target verification: Screen and sort the selected targets based on whether they are drug concentration-dependent; through comprehensive analysis of drug structure and target function, confirm Fructose-bisphosphate aldolase as the target, and take corresponding verification measures.

2. The application of shikonin target sites screened based on LIP-MS technology as claimed in claim 1, characterized in that: The Fructose-bisphosphate aldolase target and shikonin directly act against fungi.

3. The target of shikonin screened based on LIP-MS technology according to claim 1, characterized in that: The mass spectrometry analysis used an ultra-high performance liquid chromatography-mass spectrometry system nano-UPLC-MS, model: Ekspert nano LC 400 and Triple-TOF 5600+, AB Sciex, which separated peptides on a ChromXP C18 column at a flow rate of 5 μl / min; the peptides were eluted using a 90-minute acetonitrile-water gradient.

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