A method for screening a shikonin acting target point based on LIP-MS technology

Abstract

The application discloses a method for screening shikonin acting target points based on LIP-MS technology, and relates to the technical field of medicines, and has the technical features that the application adopts a widely specific protease to perform limited enzymolysis on a protein sample, utilizes the characteristic that drug and target protein combination can improve the conformation stability of the combination region peptide segment, further uses trypsin to perform secondary enzymolysis, collects a peptide fingerprint by using quantitative proteomics technology, analyzes and compares the changes of the peptide fingerprints of a control group and a drug treatment group, screens out the specificity of the 'conformational retention peptide segment' after drug treatment, identifies the target protein information corresponding to the peptide segment by database matching, compared with the traditional DARTS method, the non-labeled drug target analysis based on the peptide fingerprint changes the detection object from the protein into the peptide segment, and is expected to characterize the low-affinity multi-target drug and protein interaction, low-abundance protein, large molecular weight protein local conformation change information, so as to improve the accuracy and sensitivity of target analysis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and specifically to a method for screening shikonin targets based on LIP-MS technology. Background Technology

[0002] Shikonin (SK) is a fat-soluble naphthoquinone compound extracted from the traditional Chinese medicine *Lithospermum erythrorhizon*. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, antiviral, and antifungal effects. Studies have shown that shikonin exhibits strong antifungal activity against *Candida albicans*, particularly against fluconazole-resistant fungi. Combining shikonin with azoles and polyenes can reduce the dosage of these drugs and restore their antifungal activity against drug-resistant fungi, making it a good antifungal synergist. However, the specific targets that shikonin directly interacts with in *Candida albicans* cells, and the mechanisms by which it regulates upstream and downstream genes or pathways to exert its antifungal effect, remain unanswered questions.

[0003] Drug targets are the key biological basis for drug activity, and their discovery and identification have always been a research hotspot in various fields of pharmacy. In recent years, various labeled and unlabeled drug target analysis methods have been developed. Relatively speaking, unlabeled drug target identification significantly shortens the target analysis time because it eliminates the need for structure-activity relationship and tag modification studies of small molecules, thus attracting in-depth research and widespread application. Unlabeled drug target identification methods mainly include DARTS (Drug Affinity Response Target Stability Analysis), SPROX (Oxidation Rate-Based Protein Stability Assessment), and CETSA (Cellular Thermal Shift Analysis). These methods identify drug targets by observing changes in biophysical properties (such as stability, oxidation rate, or thermal stability) resulting from drug binding to target proteins. However, these methods also have certain limitations. First, DARTS technology has limited ability to detect low-abundance targets, and the hydrolytic sensitivity of the target protein and the specificity of the selected protease may affect the results. Second, SPROX technology is limited by the methionine content in the protein and is currently mainly applicable to the analysis of cell lysates, and cannot be directly applied to live cell systems. In addition, CETSA technology requires high drug concentrations, is time-consuming and costly, and lacks information on the binding of drugs to specific domains of target proteins.

[0004] Therefore, the present invention aims to provide a method for screening shikonin targets based on LIP-MS technology to solve the above-mentioned problems. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide a method for screening the target of shikonin based on LIP-MS technology. The method uses a label-free drug target analysis method based on peptide fingerprinting to extract total protein from Candida albicans cells under shikonin intervention, performs restriction enzymatic digestion with Proteinase K, compares the changes in peptide fingerprinting between the control group and the drug-treated group, screens out specific "conformation-maintaining peptides" in the shikonin-treated group, and identifies the target protein of shikonin that inhibits Candida albicans biofilm through database matching.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for screening shikonin targets based on LIP-MS technology, the technique comprising the following steps:

[0008] S1. Protein extraction: Monoclonal Candida albicans was inoculated into 1 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 16 hours. After the culture was completed, the cells were washed three times with PBS, and 5 mL of protein extraction buffer was added. The mixture was then mixed with twice the volume of acidified glass beads and extracted under ice bath conditions. The protein concentration was detected using a BCA protein concentration assay kit and diluted with PBS to 1.0 mg / mL. The protein was then stored at -80℃ for later use.

[0009] S2. Limited protein hydrolysis: Take 100 µg of diluted protein solution, add appropriate concentration of drug or DMSO, and add proteinase K to the sample at an enzyme / substrate ratio of 1:100. Incubate the sample for 3 minutes, then heat to inactivate PK. Subsequently, 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 reduced cysteine ​​residues. Add trypsin at an enzyme / substrate ratio of wt / wt 1:100 and incubate overnight at 37°C. Stop digestion and precipitate sodium deoxycholate by adding 98% vol / vol formic acid to a final concentration of 2% vol / vol. Finally, perform high-speed digestion for 10 minutes to remove the precipitate and desalt the sample. 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, models: Ekspert nano LC 400 and Triple-TOF 5600+, AB Sciex, USA). The system was used on a ChromXP C18 column (0.3 mm × 150 mm, 3 µm, AB Sciex, USA) at a flow rate of 5 µl / min to separate peptides. Elution of peptides was performed using a 90-minute acetonitrile-water gradient. Quantification of the Candida albicans proteome was achieved using SWATH-based mass spectrometry; data acquisition and analysis were performed using Analyst® 1.7 software and Protein Pilot 4.5; Markerview software and the Uniprot database were used, and a differential protein information table was established using the Uniprot database; by comparing the differences in effective peptides, potential targets of shikonin for Candida albicans were screened.

[0011] S4. Target Validation: Screened targets are ranked and prioritized based on their drug concentration dependence; through comprehensive analysis of drug structure and target function, Fructose-bisphosphate aldolase is confirmed as the target, and corresponding validation measures are taken.

[0012] The fructose-bisphosphate aldolase target interacts directly with shikonin to fight fungi.

[0013] The beneficial effects of this plan are:

[0014] This invention proposes a label-free drug target analysis method based on peptide fingerprinting. Total protein from *Candida albicans* cells treated with *Lithospermum erythrorhizon* is extracted, and proteinase K is used for restriction digestion. Changes in peptide fingerprints between the control and drug-treated groups are compared to screen for specific "conformation-maintaining peptides" in the *Lithospermum erythrorhizon* treatment group. Database matching is then used to identify the target proteins for *Lithospermum erythrorhizon* biofilm inhibition by *Lithospermum erythrorhizon*. Compared to the traditional DARTS method, label-free drug target analysis based on peptide fingerprinting transforms the detection target from proteins to peptides, potentially characterizing low-affinity multi-target drug-protein interactions, low-abundance proteins, and local conformational changes in high-molecular-weight proteins, thereby improving the accuracy and sensitivity of target analysis. Attached Figure Description

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

[0016] Figure 2 This is an affinity measurement diagram of SK and the potential target Fba1 in an embodiment of the present invention;

[0017] Figure 3 In this embodiment of the invention, SK inhibits the activity of Fba1 (SK concentration 0-32µg / ml).

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

[0019] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0021] Example:

[0022] The present invention provides a method for screening shikonin targets based on LIP-MS technology, as described above, comprising the following steps:

[0023] S1. Protein extraction: Monoclonal Candida albicans was inoculated into 1 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 16 hours. After the culture was completed, the cells were washed three times with PBS, and 5 mL of protein extraction buffer was added. The mixture was then mixed with twice the volume of acidified glass beads and extracted under ice bath conditions. The protein concentration was detected using a BCA protein concentration assay kit and diluted with PBS to 1.0 mg / mL. The protein was then stored at -80℃ for later use.

[0024] S2. Limited protein hydrolysis: Take 100 µg of diluted protein solution and add an appropriate concentration of drug or DMSO. Add proteinase K to the sample at an enzyme / substrate ratio of 1:100 and incubate for 3 minutes. Then heat to inactivate PK. Subsequently, 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 reduced cysteine ​​residues. Add trypsin at an enzyme / substrate ratio (wt / wt) of 1:100 and incubate overnight at 37°C. Stop digestion and precipitate sodium deoxycholate by adding 98% (vol / vol) formic acid to a final concentration of 2% (vol / vol). Finally, desalt the sample for 10 minutes at high speed to remove the precipitate. 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, models: Ekspert nano LC 400 and Triple-TOF 5600+, AB Sciex, USA). The system was used on a ChromXP C18 column (0.3 mm × 150 mm, 3 µm, AB Sciex, USA) at a flow rate of 5 µl / min to separate peptides. Elution of peptides was performed using a 90-minute acetonitrile-water gradient. Quantification of the Candida albicans proteome was achieved using SWATH-based mass spectrometry; data acquisition and analysis were performed using Analyst® 1.7 software and Protein Pilot 4.5; Markerview software and the Uniprot database were used, and a differential protein information table was established using the Uniprot database; by comparing the differences in effective peptides, potential targets of shikonin for Candida albicans were screened, such as... Figure 1 As shown;

[0026] S4. Target Validation: The screened potential targets 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). Based on a comprehensive analysis of drug structure and target function, Fructose-bisphosphate aldolase (Fba1) was considered to be one of the most likely targets for SK to inhibit Candida albicans, and corresponding validation was subsequently carried out.

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

[0028]

[0029] Validation Analysis:

[0030] SK exhibits specific binding activity to Fba1:

[0031] Surface plasmon resonance (SPR) analysis was used to quantitatively assess the binding affinity of SK to Fba1. The affinity constant (KD) between SK and Fba1 was determined using a Biacore T200 instrument, and the results are as follows: Figure 2 As shown. Vertical lines indicate a size of 5.10 × 10. -7 MKD's affinity for bonding.

[0032] SK inhibits Fba1 activity:

[0033] To test whether the interaction between SK and Fba1 affects Fba1 function, the inhibitory effect of SK on the activity of fructose-2-bisphosphate aldolase (Fba1) was investigated. The results are as follows: Figure 3 As shown, the half-maximal inhibitory concentration (IC50) of SK for inhibiting Fba1 activity was 4.639 ± 0.253 µg / ml.

[0034] Inhibitory effect of SK on mycelia:

[0035] To investigate whether the inhibitory effect of SK on mycelial growth is related to Fba1, the differences in the effects of SK on wild-type fungi and Fba1 single-arm knockout fungi were compared. The results are as follows: Figure 4 As shown, the results indicate that, compared to the wild-type strain, the Fba1 single-arm knockout strain exhibited more significant growth inhibition under the influence of SK. This finding suggests that the presence or activity of Fba1 is crucial for alleviating the inhibitory effect of SK. Specifically, Fba1 may serve as a direct or indirect target of SK, participating in the regulation of 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 intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for screening shikonin targets based on LIP-MS technology, characterized in that: The technology includes the following steps: S1. Protein extraction: Monoclonal Candida albicans were inoculated into 1 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 16 hours. After the culture was completed, the cells were washed three times with PBS, and 5 mL of protein extraction buffer was added. The mixture was then mixed with twice the volume of acidified glass beads and extracted under ice bath conditions. The protein concentration was detected using a BCA protein concentration assay kit and diluted with PBS to 1.0 mg / mL. The protein was then stored at -80℃ for later use. S2. Limited protein hydrolysis: Take 100 µg of diluted protein solution, add appropriate concentration of drug or DMSO, and add proteinase K to the sample at an enzyme / substrate ratio of 1:

100. Incubate the sample for 3 minutes, then heat to inactivate PK. Subsequently, 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 reduced cysteine ​​residues. Add trypsin at an enzyme / substrate ratio of wt / wt 1:100 and incubate overnight at 37°C. Stop digestion and precipitate sodium deoxycholate by adding 98% vol / vol formic acid to a final concentration of 2% vol / vol. Finally, centrifuge at 16000g for 10 minutes to remove the precipitate, and after desalting, transfer the supernatant for mass spectrometry analysis. S3. Mass Spectrometry Analysis: Quantification of the Candida albicans proteome was achieved using SWATH-based mass spectrometry; data acquisition and analysis were performed using Analyst® 1.7 software and Protein Pilot 4.5; Markerview software and the Uniprot database were used to establish a differential protein information table; by comparing the differences in effective peptides, potential targets of shikonin for Candida albicans were screened. S4. Target Validation: Screened targets are ranked and prioritized based on their drug concentration dependence; through comprehensive analysis of drug structure and target function, Fructose-bisphosphate aldolase is confirmed as the target, and corresponding validation measures are taken.

2. The method for screening shikonin targets based on LIP-MS technology as described in claim 1, characterized in that: The fructose-bisphosphate aldolase target interacts directly with shikonin to fight fungi.

3. The method for screening shikonin targets based on LIP-MS technology as described in claim 1, characterized in that: The mass spectrometry analysis was performed using an ultra-high performance liquid chromatography-mass spectrometry system, nano-UPLC-MS, model: Ekspert nano LC400 and Triple-TOF 5600+, AB Sciex. The system separated peptides on a ChromXP C18 column at a flow rate of 5 µl / min; the peptides were eluted using an acetonitrile-water gradient for 90 minutes.

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