A triene mycin photoaffinity probe, preparation method and application thereof

Abstract

The application discloses a trienomycin photoaffinity probe, a preparation method and application, and comprises the following steps: taking Trienomycinol as a substrate, carrying out a substitution reaction with an iodine-substituted bisaziridine, then removing propylidene protection to obtain a corresponding photoaffinity negative probe; using Fmoc-D-alanine to selectively esterify a hydroxyl group at position 11 of the negative probe, then removing an Fmoc protection group on nitrogen and carrying out condensation with cyclohexanecarboxylic acid to obtain a corresponding photoaffinity positive probe; trienomycin protein target identification comprises the following steps: (1) enrichment of an intermediate Trienomycinol and synthesis of a trienomycin photoaffinity positive probe; (2) target identification of trienomycin by using the photoaffinity probe and molecular biology research on the target. The separated trienomycin intermediate, the synthesized trienomycin probe derivative and the identified trienomycin protein target have the advantages of high originality and high yield.

Description

Technical Field

[0001] This invention belongs to the field of chemical biology, specifically a trienemycin photoaffinity probe, its preparation method, and its application. Background Technology

[0002] Trienomycin A (TA) is a macrocyclic natural product. Chemically, it is a 21-membered macrocyclic lactam with chiral carbons at positions 3, 11, and 13, and contains three consecutive E-type double bonds, hence its name. Trienomycin A was first isolated from Streptomyces. Streptamyces In sp. No. 83-16, scientists from China and abroad subsequently isolated it from different Streptomyces, such as Streptomyces USF-319, Streptomyces sp. 91614、 Streptomyces sp. PAE37, Streptomyces seoulensis IFB-A01 and Streptomyces cacaoi subsp. asoensis H2S5. This Streptomyces strain H2S5 was collected by Professor Gao Jinming's research group at Northwest A&F University in the Qinling Mountains of Shaanxi Province. Its acquisition channels are different from those of the Streptomyces mentioned above, which reflects the biodiversity and intergeneric relationships of Streptomyces.

[0003] Secondary metabolites of Streptomyces are the main raw materials for obtaining trienemycin. Apart from isolation from Streptomyces secondary metabolites, other routes cannot be scaled up. The following is a list of relevant literature on the isolation of natural products from Streptomyces.

[0004] Besides the separation methods from natural products, there are also related synthetic reports both domestically and internationally. Although synthesis is also a method for obtaining trienemycin, it is inefficient, time-consuming, and has a low yield, failing to meet the needs of routine trienemycin research.

[0005] In recent years, scientific research on trienomycin A has attracted widespread attention both domestically and internationally, driving the development of related natural products (antibiotic-type natural products). Currently, domestic research progress is slow. Traditional natural product isolation methods alone cannot meet the content requirements of research and the market for trienomycin A, mainly because natural trienomycin A is difficult to obtain and its sources are very limited. Traditional separation and extraction processes are also costly, and synthetic methods are not yet mature. To address the shortage of trienomycin A sources, it is hoped that optimized methods can be used to obtain trienomycin A on a large scale, thus meeting subsequent research and market demands. Summary of the Invention

[0006] The purpose of this invention is to provide a trienemycin photoaffinity probe, its preparation method and application, a scheme for isolating key intermediates of trienemycin, the synthesis of trienemycin photoaffinity small molecule probes and derivatives, the identification of direct targets of trienemycin, and the synthesis study of simplified trienemycin derivatives.

[0007] A trienemycin photoaffinity probe comprises: using Trienomycinol as a substrate, reacting it with iodinated diazinonyl esters in a substitution reaction, and then removing the propionyl protection to obtain the corresponding photoaffinity-negative probe;

[0008] The corresponding photoaffinity positive probe was obtained by selectively esterifying Fmoc-D-alanine with the hydroxyl group at position 11 of the negative probe, removing the Fmoc protecting group on the nitrogen atom, and then condensing it with cyclohexane.

[0009] Optionally, the preparation of the photoaffinity-negative probe specifically includes: Trienomycinol was dissolved in acetone and stirred evenly with 2,2-dimethoxypropane. At room temperature, a catalytic amount of racemic camphor sulfonic acid was added and stirred evenly. After the reactants were completely reacted, triethylamine was added and stirred at room temperature. The resulting liquid was evaporated under reduced pressure to obtain a crude product, which was then extracted with an organic solvent and dried to obtain compound 1. The molar ratio of Trienomycinol to racemic camphor sulfonic acid was 1:0.01. Compound 1 and potassium carbonate were reacted with N,N-dimethylformamide and acetone at room temperature under an argon atmosphere and stirred. Then 3-(3-yne-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was added and stirred until homogeneous. The mixture was then sealed in the dark and reacted at 40 °C for 72 h. Compound 2 was obtained after extraction with an organic solvent and drying. The molar ratio of compound 1:potassium carbonate:3-(3-yne-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was 1:5:5. After compound 2 was dissolved in methanol, a catalytic amount of racemic camphor sulfonic acid was added, and the mixture was sealed and stirred for about 1.5 h after being protected from light by paper. After the reaction of the raw materials was complete, triethylamine was added and stirred at room temperature for 30 min. On a molar ratio basis, compound 2: racemic camphor sulfonic acid = 1:0.01.

[0010] Optionally, Fmoc-D-alanine is selectively esterified with the hydroxyl group at position 11 of the negative probe, followed by removal of the Fmoc protecting group on the nitrogen atom and condensation with cyclohexanecarboxylic acid to obtain the corresponding photoaffinity positive probe, specifically including: Fmoc-D-alanine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalytic amount of 4-dimethylpyridine were added to dichloromethane and stirred until homogeneous at 0 °C under an argon atmosphere. After adding N,N-diisopropylethylamine and stirring until homogeneous, a negative probe was added. The mixture was then sealed and stirred in the dark to obtain compound 3. Molar ratio meter, photoaffinity-negative probe: Fmoc-D-alanine: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:5; Compound 3 was added to N,N-dimethylformamide at -40°C under an argon atmosphere and stirred until homogeneous; then a tetrahydrofuran solution of tetrabutylammonium fluoride was slowly added. After the addition of the compound was completed, the reaction was carried out in the dark to obtain compound 4; the molar ratio of compound 3 to tetrabutylammonium fluoride was 1:5. Cyclohexane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and a catalytic amount of 4-dimethylpyridine were added to dichloromethane and stirred until homogeneous at -20 °C under an argon atmosphere; N,N-diisopropylethylamine was added and stirred until homogeneous, and then compound 4 from the previous step was added, and the reaction was carried out in the dark. On a molar ratio basis, compound 4: cyclohexanecarboxylic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 1-hydroxybenzotriazole: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:5.

[0011] Optionally, the Trienomycinol is obtained from the fermentation product of strain H2S5 by column chromatography, lithium aluminum hydride reduction and gel purification to obtain the intermediate Trienomycinol with a purity of more than 80%.

[0012] Optional, the specific process includes: Enrichment of secondary metabolites of strain H2S5: The H2S5 strain was inoculated into a culture flask containing SPY medium and cultured in a constant temperature shaker at 27°C and 120 rpm for 7 days. After the culture was completed, the bacterial cells and bacterial solution were separated. The bacterial solution was extracted three times with 50 L of ethyl acetate, and the organic phase was concentrated to finally obtain the extract. Separation of Trienomycinol: The extract was mixed with 200-mesh normal silica gel and eluted with ethyl acetate and dichloromethane:methanol = 10:1 as the eluent. The extract was detected by TLC, and the blue portion of the vanillin reagent was collected and the eluent was concentrated. After drying the concentrate, it was reacted with lithium aluminum hydride in tetrahydrofuran in an anhydrous and oxygen-free system. After the reaction of the raw materials was complete, the reaction was quenched at 0°C with a saturated potassium sodium tartrate solution and stirred overnight at room temperature. Then, it was extracted three times with ethyl acetate, the organic phases were combined, washed and dried, and the resulting liquid was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography under gradient elution conditions from dichloromethane:methanol = 50:1 to dichloromethane:methanol = 10:1 by volume to obtain crude Trienomycinol. The crude Trienomycinol was purified by methanol gel column chromatography.

[0013] The preparation method of any of the trienycin photoaffinity probes of the present invention includes: using Trienomycinol as a substrate, reacting it with iodopropyl diazinon via a substitution reaction, and then removing the propylidene protection to obtain the corresponding photoaffinity-negative probe; specifically: dissolving Trienomycinol in acetone, stirring it evenly with 2,2-dimethoxypropane, adding a catalytic amount of racemic camphor sulfonic acid at room temperature and stirring evenly, and after the raw material reaction is complete, adding triethylamine and stirring at room temperature; the resulting liquid is evaporated under reduced pressure to obtain a crude product, which is then extracted with an organic solvent and dried to obtain compound 1; the molar ratio of Trienomycinol:racemic camphor sulfonic acid = 1:0.01; compound 1 and potassium carbonate are added to N,N-dimethylformamide and acetone at room temperature under an argon atmosphere and stirred; then 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine is added and stirred evenly; after being protected from light and sealed, the reaction is continued at 40 °C for 72 days. h; Compound 2 was obtained after extraction and drying with organic solvent; the molar ratio of compound 1: potassium carbonate: 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was 1:5:5; after compound 2 was dissolved in methanol, a catalytic amount of racemic camphor sulfonic acid was added, and the mixture was sealed and stirred for about 1.5 h after being protected from light by paper; after the reaction of the raw materials was complete, triethylamine was added and stirred at room temperature for 30 min; the molar ratio of compound 2: racemic camphor sulfonic acid was 1:0.01; Selective esterification of Fmoc-D-alanine with the 11-hydroxyl group of the negative probe, followed by removal of the nitrogen-containing Fmoc protecting group and condensation with cyclohexanecarboxylic acid, yields the corresponding photoaffinity positive probe. Specifically, the probe consists of Fmoc-D-alanine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalytic amount of 4-dimethylpyridine, at 0... At -40°C under an argon atmosphere, dichloromethane was added and stirred until homogeneous. N,N-diisopropylethylamine was added and stirred until homogeneous, followed by the addition of a negative probe. After light protection, the reaction was sealed and stirred to obtain compound 3. The molar ratio of photoaffinity negative probe:Fmoc-D-alanine:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride:4-dimethylpyridine:N,N-diisopropylethylamine was 1:2:2:0.01:5. Compound 3 was then reacted with N,N-dimethylformamide at -40°C under an argon atmosphere. The mixture was stirred until homogeneous. Then, a tetrahydrofuran solution of tetrabutylammonium fluoride was slowly added. After the addition was complete, the reaction was performed in the dark to obtain compound 4. The molar ratio of compound 3:tetrabutylammonium fluoride was 1:5. Cyclohexane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and a catalytic amount of 4-dimethylpyridine were reacted at -20°C. At ℃, under an argon atmosphere, dichloromethane was added and stirred until homogeneous; N,N-diisopropylethylamine was added and stirred until homogeneous, then compound 4 from the previous step was added, and the reaction was carried out in the dark; on a molar ratio, compound 4: cyclohexanecarboxylic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 1-hydroxybenzotriazole: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:5.

[0014] The application of any of the trienemycin photoaffinity probes described in this invention for the identification of trienemycin targets.

[0015] Optionally, it identifies targets using limited enzyme digestion mass spectrometry (LiP-MS) and narrows the scope using network pharmacology, for example, directly identifying 34 target proteins that directly bind to TA, and then narrowing the scope to 15 target proteins using network pharmacology.

[0016] Features and advantages of this invention: High reaction yield: The yield of each step in the preparation of intermediates is significantly improved compared to previous patents and literature, with some intermediates achieving yields of over 90%. High originality of invention: This invention is the first to improve existing separation processes, enabling the production of more products in a shorter time; it is the first to synthesize a small-scale photoaffinity probe for trienemycin, and the direct binding target of trienemycin was identified using this probe; in the identification of the trienemycin protein target, two identification techniques were used, one for identification and one for verification, making the results more reliable. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 This is the 1H NMR spectrum of the positive probe of this invention; Figure 2 This is the carbon NMR spectrum of the positive probe of this invention; Figure 3 This is the high-resolution mass spectrometry of the positive probe of the present invention; Figure 4 This is a traditional separation and purification process; Figure 5 For the improved separation and purification process; Figure 6 The IC50 of the positive probe in the PANC-1 cell line; Figure 7 This is a limited enzyme digestion mass spectrometry technique route; Figure 8 These are proteins identified using limited enzyme digestion mass spectrometry. Figure 9 Intersection proteins between the identified proteins and those obtained from network pharmacology; Figure 10 The peptide corresponding to the target protein HSPA5; Figure 11 The HSPA5 peptide identified by limited enzyme digestion mass spectrometry; Figure 12 The following are kinetic simulation data for the heat shock protein family: A is data for HSP90AB1; B is data for HSPA2; C is data for HSPA5; D is data for HSPA8. Figure 13 High-resolution mass spectrometry of biotin probes; Figure 14 The results are from the PullDown experiment; Figure 15 Protein thermal shift experiments for HSPA2, HSPA5, and HSP90AB1: A and B are HSPA2; C and D are HSPA5; E and F are HSP90AB1. Figure 16 Transcriptional studies of HSP90AB1, HSPA2, HSPA5 and HSPA8 proteins: A is in PANC1; B is in MIAPaCa-2; Figure 17 Changes in protein expression levels of HSPA2, HSPA5, and HSP90AB1: A and B represent HSPA2; C and D represent HSPA5; E and F represent HSP90AB1. Figure 18 Protein expression levels under the influence of CHX: A and B are HSPA2; C and D are HSPA5; E and F are HSP90AB1. Figure 19 The degradation pathways of heat shock family proteins are as follows: A and B are HSPA2; C and D are HSPA5; E and F are HSP90AB1. Figure 20 For the scratch test: A is 0h; B is 6h; C is 12h; D is 24h. Detailed Implementation

[0018] This invention relates to separation methods, derivative probe synthesis and target identification, belonging to the fields of natural product separation, chemical synthesis technology and chemical biology, and particularly to selective esterification reactions. It utilizes LiP-MS technology to identify the direct binding target of trienycin, specifically the separation of key intermediates of trienycin, the synthesis of trienycin derivative probes and the identification of trienycin protein targets.

[0019] (1) Enrichment of intermediate Trienomycinol and synthesis of trienemycin photoaffinity positive probe Trienomycinol, an intermediate with a purity of over 80%, was obtained from the fermentation product of strain H2S5 by column chromatography, lithium aluminum hydride reduction, and gel permeation purification. Trienomycinol was then substituted with iodinated diacylpropionyl, followed by deprotection of the propylidene group to yield the corresponding photoaffinity-negative probe. Subsequently, Fmoc-D-alanine was selectively esterified with the 11-hydroxyl group of the negative probe, followed by removal of the nitrogen-containing Fmoc protecting group and condensation with cyclohexanecarboxylic acid to obtain the corresponding photoaffinity-positive probe. Furthermore, the synthesis of a novel diacylpropionyl photoaffinity probe was investigated, providing theoretical and experimental support for the synthesis of novel probes.

[0020] (2) Identify the target of trienemycin using photoaffinity probes and conduct molecular biological studies on the target. First, the positive probe demonstrated significant cytotoxicity against pancreatic cancer cells PANC-1 using the MTT assay, with an IC50 value of 0.28 μM, confirming the rationality of the probe design. Second, limited-enzyme digestion mass spectrometry (LiP-MS) identified 34 target proteins that directly bind to the TA, which were then narrowed down to 15 target proteins using network pharmacology. Finally, based on the group's research on endoplasmic reticulum stress, future research on the target protein family will focus on heat shock proteins.

[0021] GO enrichment analysis revealed that trienemycin affects cellular processes, biological regulation, catalytic activity, and organelles. KEGG pathway enrichment analysis showed that differentially expressed proteins were highly enriched in cancer pathways. Subsequently, ABPP technology, cellular thermal shift, and molecular dynamics simulations verified that HSPA2, HSPA5, HSPA8, and HSP90AB1 are direct binding targets of TA, and the binding energies of trienemycin to these targets were obtained as -9.6 kcal / mol, -10.1 kcal / mol, -7.5 kcal / mol, and -12.0 kcal / mol, respectively.

[0022] qPCR studies revealed that trienzyme increased the transcriptional levels of some heat shock family proteins, while Western blotting showed that trienzyme decreased the expression levels of these heat shock proteins. This indicates that trienzyme regulates heat shock family proteins not through transcription, but by promoting their degradation and thus reducing their expression levels. Further research found that HSP90AB1 is degraded via the proteasome pathway, while HSPA2 and HSPA5 may be degraded via other pathways, such as the lysosomal pathway. HSPA5 is a key protein among these heat shock proteins; therefore, scratch assays were performed on pancreatic cancer cells overexpressing HSPA5. The results confirmed that trienzyme can inhibit cancer cell invasion and migration by downregulating HSPA5 expression.

[0023] (1) Design and synthesis of photoaffinity-tagged small molecule probes for trienemycin The purpose of this invention (1) is to use Streptomyces cacaoi subsp. asoensis H2S5 as raw material to conduct subsequent research by enriching the secondary metabolites produced by this strain.

[0024] Combination Figure 4 In the enrichment of secondary metabolites, this invention first employs a traditional separation process: after restoring the bacterial strain frozen at -80℃ to room temperature, the strain is transferred to pre-sterilized TSB medium in a clean bench and activated for 3 days in a 26℃ incubator. Subsequently, it undergoes two expansion cultures using sterilized SPY medium (26℃ shaker fermentation). The fermentation broth is concentrated after solid-liquid separation with gauze, and then subjected to four-round extraction with ethyl acetate, dissolution with methanol, and defatting with petroleum ether to obtain an extract. The 70%-90% methanol eluent (vanillin-sulfuric acid ethanol showing a blue fraction) is collected fractionally by medium-pressure chromatography, and then separated by gel chromatography, reversed-phase column chromatography, and HPLC to obtain ansamycin-like products, primarily trienycinol. Finally, trace components are reduced with lithium aluminum hydride to obtain a crude sample with Trienycinol as the main component. The entire process takes 33.5 days, which is significantly inefficient.

[0025] Combination Figure 5 To address the aforementioned issues, this invention systematically optimizes the separation process: (1) the time-consuming concentration step is eliminated, and the 60 L fermentation broth is directly extracted three times with ethyl acetate, reducing the time by more than half; (2) based on the goal of directional separation of macrocyclic lactam skeleton compounds, the medium-pressure segmentation is abandoned, and forward elution is used to effectively remove impurities (20 g of extract is concentrated to less than 5 g after processing); (3) the lithium aluminum hydride reduction step is retained, and Trienimycinol with a liquid phase purity of 98% can be obtained by gel chromatography purification. Experiments show that although omitting gel chromatography can obtain higher purity products by HPLC, due to residual small molecule impurities (estimated purity of only 60%) and longer preparation time, the scheme of gel chromatography combined with lithium aluminum hydride reduction is chosen. In addition, it was found during the fermentation process in large tanks that the metabolites of 300 L industrial fermentation are different from those of shaker fermentation, so shaker fermentation is used for subsequent cultivation and fermentation.

[0026] ; After obtaining Trienimycinol in batches, this invention proceeded with the synthesis of trienimycin photoaffinity probes.

[0027] The synthesis process of the 22-position photoaffinity-tagged negative probe is as follows: First, Trienomycinol is protected with propylene under the catalysis of camphorsulfonic acid to obtain compound 1-1; then compound 1-1 undergoes a substitution reaction with 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine to obtain compound 1-2; compound 1-2 is then deprotected with propylene to obtain the negative probe.

[0028] ; Based on the successful synthesis of the negative probe, this invention further synthesizes the positive probe. The negative probe, at a concentration of 1 mg / mL, achieves selective esterification of the 11-hydroxyl group by precisely controlling the reaction conditions, yielding compound 4; compound 4 is deprotected from Fmoc in TABF and then condensed with cyclohexanecarboxylic acid to obtain the positive probe.

[0029] .

[0030] (2) Identification of the target and mechanism of action of trienemycin The purpose of this invention (2) is to identify the target of trienemycin and study its mechanism based on the probe synthesized in (1).

[0031] PANC-1 and MIA PaCa-2 are two commonly used cell models in pancreatic cancer research. According to studies by the Mulvihill group (Deer et al., 2010) and the Henshall group, the KRAS G12D mutation in the PANC-1 cell line is widespread in pancreatic cancer, while the KRAS G12C mutation in the MIA PaCa-2 cell line is relatively rare in pancreatic cancer. Therefore, using PANC-1 as the research subject can better reflect the general pathological characteristics of pancreatic cancer.

[0032] The cytotoxicity of the positive probe against pancreatic cancer cells PANC-1 was detected by the MTT assay. The results and IC50 values ​​were obtained.

[0033] Combination Figure 6 The positive probe showed an IC50 value of 0.28 μM in the PANC-1 cell line, while the IC50 value of trienzyme in the same cell line was 0.4 μM. The positive probe exhibited cytotoxicity similar to, and slightly superior to, trienzyme. This indicates that the introduction of the diazidopropylidin group preserved the antitumor activity of trienzyme without significantly affecting it. Therefore, this positive probe can be used for subsequent target identification.

[0034] The target identification process is as follows: Figure 7 As shown: First, the data in Table 1 were obtained through DIA data acquisition and analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS): Table 1. Statistical results of the number of identified peptides and proteins.

[0035] Based on the data in Table 1, the standard deviations and coefficients of variation for the corresponding proteins and peptides in each group were calculated. The specific values ​​are as follows: for the Blank group, the standard deviations and coefficients of variation for proteins and peptides were 17.31, 0.27% and 146.32, 0.32%, respectively; for the Negative group, the standard deviations and coefficients of variation for proteins and peptides were 5.13, 0.08% and 44.0, 0.098%, respectively; for the PositiveL group, the standard deviations and coefficients of variation for proteins and peptides were 7.23, 0.12% and 99.92, 0.28%, respectively; and for the PositiveH group, the standard deviations and coefficients of variation for proteins and peptides were 4.36, 0.072% and 58.53, 0.18%, respectively.

[0036] The p-values ​​of each group and the Blank group were calculated using the T-test. The p-value of Blank group vs Negative group was 0.20, indicating no significant difference; the p-value of Blank group vs PositiveL group was 0.0024, indicating a significant difference; and the p-value of Blank group vs PositiveH group was 0.00047, indicating a significant difference.

[0037] In the above data, the coefficients of variation for protein and peptide detection values ​​within each group were all below 0.32%, indicating good data repeatability. The T-test results showed no significant difference between the Negative group and the Blank group (p = 0.20), while the PositiveL group (p = 0.0024) and the PositiveH group (p = 0.00047) showed significant differences from the Blank group. These results validate the rationality of the experimental grouping and the scientific validity of the dosage design. Through the analysis of the differentially expressed peptides, a series of differentially expressed proteins were identified, such as... Figure 8 As shown: The specific differentially expressed proteins obtained from the comparison of each group are shown in Table 2: Table 2. Differentially identified proteins

[0038] Thirty-four tristyromycin target proteins were identified using LiP-MS technology, and 14,057 pancreatic cancer-related proteins were obtained from the GeneCard database. In this invention, proteins with a correlation value greater than 15 were selected, and their intersection with the identified targets was calculated.

[0039] Combination Figure 9 The intersection of the drug target and disease-related proteins was found to be 15: GAPDH; HSPA5; HSPA8; ANXA1; HSP90AB1; LMNA; ITGB1; ANXA2; ANXA5; HSPD1; PKM; EEF1G; HNRNPK; PRDX1; and PARK7. Previous research found that the mechanism by which tris(2-tri ...

[0040] The present invention will be further illustrated by the following examples, but the present invention is not limited to the examples described. Any solvent that does not depart from the spirit of the present invention should be within the scope of protection of the present invention. Unless otherwise specified, the ratios between solvents in the present invention are volume ratios.

[0041] Example 1: Enrichment of secondary metabolites of strain H2S5: The H2S5 strain inoculum, frozen at -80℃, was transferred to a clean bench and thawed at room temperature. The inoculum was then inoculated into two gel plates. The growth of the strain was observed until activation was complete. In the clean bench, the activated strain was inoculated into an Erlenmeyer flask containing 200 mL of TSB medium. The flask was incubated at 27℃ and 120 rpm for 3 days. After incubation, the strain from the TSB medium was inoculated into a flask containing SPY medium and incubated at 27℃ and 120 rpm for 7 days. After incubation, the bacterial cells and culture were separated using gauze. The culture was extracted three times with 50 L of ethyl acetate, and the organic phase was concentrated using a thin-film evaporator to obtain the final extract.

[0042] Separation of Trienomycinol: The extract was mixed with 200-mesh normal silica gel and eluted with ethyl acetate and dichloromethane:methanol = 10:1 as the eluent. The extract was detected by TLC, and the blue portion of the vanillin reagent was collected and the eluent was concentrated.

[0043] After drying, the concentrate was reacted with 5 equiv of lithium aluminum hydride in anhydrous tetrahydrofuran in an anhydrous and oxygen-free system. The reaction was monitored by TLC. After the reactants had reacted completely, the reaction was quenched at 0°C with a saturated sodium potassium tartrate solution and stirred overnight at room temperature. The mixture was then extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The resulting liquid was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (gradient elution from dichloromethane:methanol = 50:1 to dichloromethane:methanol = 10:1) to obtain crude Trienomycinol. The crude Trienomycinol was purified by methanol gel column chromatography to obtain Trienomycinol with HPLC purity of 80%.

[0044] Preservation of H2S5 strain: The H2S5 strain inoculum, frozen at -80℃, was transferred to a clean bench and thawed at room temperature. The inoculum was then inoculated onto two gel plates. The growth of the strain was observed, and the activated strain was inoculated into an Erlenmeyer flask containing 200 mL of TSB medium. The flask was incubated at 27℃ and 120 rpm for 24 h in a constant-temperature shaker. During this time, the inoculum and a prepared mixture of 50% glycerol and water were sterilized. After incubation, 700 μL of Streptomyces culture was mixed with 700 μL of glycerol in the clean bench and stored at -80℃.

[0045] Synthesis and characterization of negative probes: ; Synthesis of Compound 1: Trienomycinol (441 mg, 1 mmol) was added to a dried 25 mL round-bottom flask. 4 mL of anhydrous acetone was added to the substrate and stirred until completely dissolved. Then, 12 mL (10.16 g, 97.6 mmol) of 2,2-dimethoxypropane was added and stirred until homogeneous. At room temperature, a catalytic amount of racemic camphorsulfonic acid (2.32 mg, 0.01 mmol) was added to the reaction system and stirred until homogeneous. After the reactants were completely reacted as detected by TLC, 1 mL (728 mg, 7.19 mmol) of triethylamine was added and stirred for 30 min at room temperature. The resulting liquid was evaporated to dryness under reduced pressure to obtain the crude product. The crude product was then mixed with water and ethyl acetate and extracted with ethyl acetate (50 mL). 3) Combine the organic phases, wash with saturated brine, and dry with anhydrous sodium sulfate. The resulting liquid is evaporated under reduced pressure to obtain the crude product. The crude product is then separated by column chromatography (dichloromethane:methanol = 50:1) to give a white solid 1, with a yield of 90%. The molar ratio of Trienomycinol to racemic camphorsulfonic acid in the reactants is: n(Trienomycinol):n(racemic camphor sulfonic acid) =1:0.01; Compound 1: HRESIMS(positive) m / z 504.2722 [M+Na]+ (calcd forC29H39NO5Na, 504.2726). 1H NMR (400 MHz, CDCl3) δ:7.81 (s, 1H), 7.31 (s, 1H),6.51 (s, 1H), 6.25–6.17 (m, 3H), 6.05 (m, 2H), 5.89–5.79 (m, 1H), 5.60 (dd, J= 15.0, 8.0 Hz, 1H), 5.24 (t, J = 6.5, 1H), 4.57 (d, J = 5.5 Hz, 1H), 4.02(t, J = 10.6 Hz,1H), 3.56–3.48 (m, 1H), 3.34 (s, 3H), 2.90–2.84 (m, 1H), 2.60–2.41 (m, 4H), 2.21–2.08 (m, 3H), 1.92–1.84 (m, 1H), 1.73 (s, 3H), 1.35(s, 3H), 1.30 (s, 3H), 0.81 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ:168.9, 157.7, 144.5, 138.1, 135.3, 135.2, 134.9, 133.5, 132.3, 131.5, 128.9, 125.3, 112.5, 109.7, 106.0, 100.8, 79.6, 73.3, 68.6, 56.6, 45.2, 37.1, 36.3, 36.0, 29.6, 24.7, 24.0, 20.4, 12.7. Comparison with literature data confirmed that compound 1 is a propionylated product of trienomycinol.

[0046] ; Compound 1 (5 mg, 10.38 μmol) and ground anhydrous potassium carbonate (7.17 mg, 51.91 μmol, 5 equiv) were added to a dried 25 mL sealed tube. Under an argon atmosphere, 0.4 mL of anhydrous N,N-dimethylformamide and 0.1 mL of anhydrous acetone were added at room temperature and stirred for 30 min. Then, 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine (12.88 mg, 51.91 μmol, 5 equiv) was added and stirred until homogeneous. The tube was sealed with aluminum foil to protect it from light and reacted at 40 °C for 72 h. When TLC showed that the starting material was almost exhausted and no further product was formed, water was added to quench the reaction. A large amount of ethyl acetate was added to the mixture, and then the organic phase was washed with a 1:1 (v / v) aqueous solution of water and saturated brine (50 mL). 4) The organic phase was washed once more with saturated brine and dried over anhydrous sodium sulfate. The resulting liquid was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (dichloromethane:methanol = 100:1) to give a yellow oily compound 2, with a yield of 55% (82% yield of recovered feed). The molar ratio of compound 1, potassium carbonate, and 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bis(acrylidine) in the reactants was: n(Compound 1):n(Potassium carbonate):n(3-(3-yne-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine) =1:5:5; ; Compound 2 (2.5 mg, 4.16 μmol) was added to a dried 10 mL round-bottom flask. 1 mL of methanol was added at room temperature and stirred until homogeneous. Then, a catalytic amount of racemic camphor sulfonic acid was added. The flask was sealed with aluminum foil to protect it from light and stirred for approximately 1.5 h. After the reaction was complete as detected by TLC, 1 mL (728 mg, 7.19 mmol) of triethylamine was added and stirred for 30 min at room temperature. The resulting liquid was evaporated to dryness under reduced pressure to obtain the crude product. The crude product was then mixed with water and ethyl acetate. Extraction was then performed using ethyl acetate (50 mL). 3) Combine the organic phases, wash with saturated brine, and dry with anhydrous sodium sulfate. The resulting liquid is evaporated under reduced pressure to obtain the crude product. The crude product is then separated by column chromatography (dichloromethane:methanol = 50:1) to obtain a white solid negative probe, with a yield of 92%. The molar ratio of compound 2 to racemic camphorsulfonic acid in the reactants is: n(compound 2) : n(racemic camphor sulfonic acid) = 1 : 0.01; Negative probe: HRESIMS(positive) m / z: 584.3090 [M+Na]+ (calcd for C33H43N3O5Na, 584.3095). 1H NMR (400 MHz, CDCl3) δ: 7.18 (s, 1H), 6.46 (s,1H), 6.44 (s, 1H), 6.30–5.98 (m, 4H), 5.70-5.57 (m, 2H), 5.28–5.21 (m, 1H), 4.81 (d, J = 5.6 Hz, 1H), 4.15–4.09 (m, 1H), 3.83 (td, J = 6.3, 2.6 Hz, 2H), 3.79–3.71 (m, 1H), 3.40 (s, 3H), 2.71–2.62 (m, 2H), 2.58 – 2.44 (m, 4H), 2.07 (td, J = 6.3, 2.6 Hz, 2H), 2.00 (t, J = 2.6 Hz, 1H), 1.87 (t, J = 6.3 Hz, 2H), 1.79 (s, 3H), 1.78–1.70 (m, 5H), 0.99 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ: 168.3, 159.0, 144.1, 138.8, 137.6, 134.1, 133.2, 132.8, 130.7, 130.3, 129.5, 126.0, 112.2, 111.5, 104.4, 82.9, 78.3, 73.6, 70.2, 69.4, 62.7, 57.1, 43.9, 40.5, 37.6, 36.6, 33.1, 32.8, 29.3, 26.9, 20.4, 13.4, 11.0. ; Fmoc-D-alanine (1.1 mg, 3.56 μmol, 2 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.683 mg, 3.56 μmol, 2 equiv), and a catalytic amount of 4-dimethylpyridine were added to a dried 10 mL sealed tube. Under an argon atmosphere at 0 °C, 1 mL of anhydrous dichloromethane was added and stirred until homogeneous. Then, N,N-diisopropylethylamine (1.15 mg, 8.9 μmol, 1.5 μL, 5 equiv) was added and stirred until homogeneous. A negative probe (1 mg, 1.7 μmol) was added to the system, and the mixture was sealed and stirred for approximately 48 h after being protected from light with aluminum foil. TLC analysis was performed until a large amount of the starting material disappeared and no more products were formed. Water was then added to quench the reaction. The resulting mixture was extracted with dichloromethane (50 mL). 3) Combine the organic phases, wash with saturated brine, and dry with anhydrous sodium sulfate. The resulting liquid is evaporated under reduced pressure to obtain the crude product. The crude product is then separated by column chromatography (dichloromethane:methanol = 100:1) to give a white solid 3, yield 45%. The molar ratio of the photoaffinity-negative probe, Fmoc-D-alanine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylpyridine, and N,N-diisopropylethylamine in the reactants is: n(photoaffinity-negative probe):n(Fmoc-D-alanine):n(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride):n(4-dimethylpyridine):n(N,N-diisopropylethylamine) = 1:2:2:0.01:5; ; Compound 3 (5 mg, 5.85 μmol) was added to a dried 10 mL reaction tube. 1 mL of anhydrous N,N-dimethylformamide was added and stirred until homogeneous at -40°C under an argon atmosphere. After homogeneity, a 1 mol / L tetrabutylammonium fluoride solution in tetrahydrofuran (7.64 mg, 29.24 μmol, 29.24 μL, 5 equiv) was slowly added. After the compound was completely added, the reaction tube was protected from light by aluminum foil. The reaction time was approximately 4 h. TLC monitoring confirmed complete reaction of the reactants, and water was added to quench the reaction. A large amount of ethyl acetate was added to the mixture, and the organic phase was washed with a 1:1 (v / v) aqueous solution of water and saturated brine (50 mL). 4) The organic phase was washed once more with saturated brine and dried over anhydrous sodium sulfate. The resulting liquid was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (dichloromethane:methanol = 20:1) to give a white solid 4. The molar ratio of compound 3 to tetrabutylammonium fluoride in the reactants is: n(compound 3):n(tetrabutylammonium fluoride) = 1:5; ; Cyclohexane (0.76 mg, 5.93 μmol, 1.5 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mg, 5.93 μmol, 1.5 equiv), 1-hydroxybenzotriazole (0.8 mg, 5.93 μmol, 1.5 equiv), and a catalytic amount of 4-dimethylpyridine were added to a dried 10 mL reaction tube. Under an argon atmosphere at -20 °C, 1 mL of anhydrous dichloromethane was added and stirred until homogeneous. Then, N,N-diisopropylethylamine (2.5 mg, 19.75 μmol, 3.4 μL, 5 equiv) was added and stirred until homogeneous. After homogeneity, the crude product from the previous step (2.5 mg, 3.95 μmol) was added to the system, and the tube was sealed with aluminum foil to protect it from light. The reaction time was approximately 4 h. After complete reaction of the starting materials by TLC, water was added to quench the reaction. The resulting mixture was extracted with dichloromethane (50 mL). 3) Combine the organic phases, wash with saturated brine, and dry with anhydrous sodium sulfate. The resulting liquid is evaporated under reduced pressure to obtain the crude product. The crude product is then separated by column chromatography (dichloromethane:methanol = 50:1) to obtain a positive probe; the two-step yield is 60%. The molar ratio of compound 4, cyclohexanecarboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-dimethylpyridine, and N,N-diisopropylethylamine in the reactants is: n(compound 4):n(cyclohexanecarboxylic acid):n(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride):n(1-hydroxybenzotriazole):n(4-dimethylpyridine):n(N,N-diisopropylethylamine)=1:2:2:0.01:5; Positive probe: HRESIMS (positive) m / z: 743.4374 [M+H]+ (calcd for C43H59N4O7, 743.4378). 1H NMR (400 MHz, CDCl3) δ: 7.37 (s, 1H), 6.50 (s, 1H), 6.37 (s, 1H), 6.32–6.02 (m, 4H), 5.89 (d, J = 6.40 Hz, 1H), 5.68–5.52 (m, 2H), 5.18 (s, 1H), 4.97–4.88 (m, 1H), 4.64 (br. s, 1H), 4.37 (p, J = 7.0 Hz, 1H), 4.16–4.06 (m, 1H). 3.83 (m, 2H), 3.38 (s, 3H), 2.76 (m, 1H), 2.60–2.41(m, 4H), 2.37–2.18 (m, 4H), 2.17–2.02 (m, 4H), 2.00 (t, J = 2.6 Hz, 1H),1.97-2.01 (m, 1H),1.87 (t, J = 6.3 Hz, 2H), 1.83-1.90 (m, 2H)1.79 (s, 3H),1.74 (m, 3H), 1.41 (d, J = 7.5 Hz, 3H), 1.37-1.45 (m, 2H) 1.30 (m, 3H), 0.91(d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ:176.8, 173.1, 168.1, 159.0,144.2, 138.8, 138.6, 134.3, 133.9, 133.5, 131.0, 129.9, 129.6, 124.7, 112.6,111.8, 104.8, 82.9, 78.9, 75.4, 69.4, 68.6, 62.7, 56.9, 48.7, 45.1, 43.9,39.5, 36.6, 33.6, 33.1, 32.8, 29.6, 29.5, 29.4, 26.9, 25.8, 25.8, 25.7, 20.6, 17.7, 13.4, 10.1. Detailed proton, carbon, and mass spectra are attached. Figure 1 , 2 And 3.

[0047] Example 2: Cell culture: Human pancreatic cancer cells PANC-1 are adherent cells, while MIA PaCa-2 are semi-adherent cells. Both cell types were removed from liquid nitrogen (or -80°C) and rapidly thawed in a 37°C water bath. After thawing, the cells were centrifuged at 1300 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in pre-warmed (37°C) culture medium. The resuspended cells were then transferred to 6 cm culture dishes and incubated at 37°C with 5% CO2. Cell status was observed daily. PANC-1 cells adhered after approximately 12 hours, while MIA PaCa-2 cells, being semi-adherent, adhered more slowly, taking approximately 18 hours, and a small number of unadhered round cells were observed; this is normal for these cells.

[0048] Cells in the logarithmic growth phase were passaged using the following steps: First, the culture medium, trypsin, and PBS buffer were warmed in a 37°C water bath. Next, the old culture medium was discarded, and the cells were washed twice with warm PBS. The PBS was then discarded, and trypsin was added for digestion (approximately 0.4 mL of trypsin for a 6 cm culture dish, and 1 mL for a 10 cm culture dish). The digestion time was approximately 40 s for PANC-1 and approximately 1 min for MIAPaCa-2. The digestion was then stopped by adding culture medium, and the cells were aspirated from the bottom of the dish. Finally, the cell suspension was transferred to centrifuge tubes and centrifuged at 1500 rpm for 5 min. The supernatant was then discarded, and 1 mL of fresh culture medium was added and the cells were aspirated and passaged. The passage ratio was 1:2 or 1:4.

[0049] PANC-1 medium: 50 mL DMEM, 10% fetal bovine serum, 1% penicillin / streptomycin.

[0050] MIA PaCa-2 medium: 50 mL DMEM, 10% fetal bovine serum, 3.5% horse serum, 1% penicillin / streptomycin.

[0051] mRNA extraction: The cell culture process here is the same as described above. After culture, mRNA extraction is performed according to the following steps: (1) Taking a 10 cm culture dish as an example. After cell culture is completed, discard the culture medium, wash with PBS, then place the cell culture dish on ice, add 1 mL TRIzol and spread it evenly over the entire culture dish, and let it stand for 15 min; (2) Draw all the liquid into an EP tube, add 0.5 mL of chloroform, shake vigorously, and let stand on ice for 15 min; (3) Centrifuge at 1400 rcf for 15 min at 4℃. Gently mix the supernatant with 0.3 mL of isopropanol and let stand at -20℃ for 20 min; (4) Centrifuge at 4℃ and 1400 rcf for 10 min. Discard the supernatant and keep the precipitate. Wash twice with 75% ethanol (7.5 mL anhydrous ethanol + 2.5 mL DEPC water), centrifuge at 4℃ and 12000 rcf, and keep the precipitate. After each centrifugation, leave the container open for 5 min to allow the solvent to evaporate naturally.

[0052] (5) Add 30 μL of DEPC water to dissolve and measure the RNA concentration.

[0053] Western blot: (1) Cell digestion reference cell culture. Digested cells were centrifuged at 1500 rpm for 5 min. After processing, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS buffer. The above operation was repeated. After obtaining the cell pellet, it was placed on ice for later use. Approximately 60 μL of lysis buffer (60 μL of cell lysis buffer requires the addition of 60 μL Lysis Buffer, 0.6 μL of protease inhibitor, 0.6 μL of phosphatase inhibitor A, and 0.6 μL of phosphatase inhibitor B) was added to each cell pellet in a centrifuge tube, and the pellet was placed on ice for lysis. The pellet was vortexed every 5 min for approximately 1 h. After lysis, the pellet was centrifuged at 12000 rpm for 30 min at 4 °C. After centrifugation, the supernatant was carefully aspirated and placed in a new centrifuge tube. The protein concentration was detected using a BCA protein concentration assay kit. After balancing with PBS, Loading Buffer was added and the pellet was boiled in boiling water for 10 min. The pellet was then cooled on an ice box, and the protein solution was stored at -20 °C.

[0054] (2) Thaw the protein solution obtained in the previous step on an ice box. Prepare the gel using a 7.5% gel preparation kit. After gel preparation, centrifuge and vortex the sample before loading. Run the bands in the electrophoresis buffer at a constant voltage of 80 V to build up the gel, then increase the voltage to 140 V to run to the bottom. After electrophoresis, transfer the membrane to the transfer buffer. Place the gel in the transfer clamp in the following order: black side of the clamp, sponge, gel, NC membrane, sponge, colorless side of the clamp. Place the transfer clamp in the transfer tank, with the black and red sides corresponding. Transfer at 400 mA for 45 min. After transfer, stain the membrane with Ponceau S for 30 s. Wash the membrane with dd water and observe the protein bands. After washing, block in skim milk powder for 1 h. After blocking, cut the desired bands according to the marker. Incubate the bands overnight (4℃) in the corresponding primary antibody. After primary antibody incubation, the bands were washed with TBST twice for 5 minutes, followed by two washes of 10 minutes each. After washing, the bands were placed in their corresponding secondary antibodies and incubated at room temperature for 1 hour. After incubation, the bands were washed again with TBST twice for 5 minutes, followed by two washes of 10 minutes each. After washing, the developing agent was evenly applied to the bands, and the expression of each protein was recorded using a high-sensitivity multi-functional imager. ImageJ was used for statistical analysis of the grayscale values.

[0055] Plasmid transfection: Passaged PANC-1 cells were placed in 6 cm cell culture dishes and cultured further to observe cell adhesion. After cell adhesion was complete, cell count was monitored during culture. When the cells reached approximately 60% confluency, the original culture medium was discarded, and serum-free and antibiotic-free DMEM medium was added, followed by starvation culture for 4 hours. After starvation culture, the plasmid was transfected into the cells according to the transfection kit instructions, and the cells were cultured for another 6 hours. After 6 hours, the culture medium containing the plasmid was discarded, and normal PANC-1 medium was added, followed by further culture for 24 hours.

[0056] Pull-down experimental method: Twenty-four hours after transfection with the relevant plasmid, the positive probe biotin was added to the culture medium and cultured for another 6 hours. Before cell lysis, the cells were incubated at 365 nm for 25 minutes and then lysed using IP buffer. Protein quantification was then performed using a BCA kit. The quantified protein suspension was divided into two portions: 10 μL of streptavidin-containing magnetic beads were added to each 90% volume protein suspension and incubated at 4°C for 4 hours. After incubation, the magnetic beads were enriched using a magnet, the supernatant was discarded, and the beads were washed twice with PBS. The mixture was then diluted with IP buffer and Loading Buffer, boiled in water for 10 minutes, and the magnetic beads were discarded. The supernatant was stored at -20°C. The remaining 10% volume protein suspension was added to 4× Loading Buffer, boiled in water for 10 minutes, and then stored at -20°C.

[0057] LiP-MS target identification process: This invention employs a combined LiP-MS and ABPP approach to enhance the accuracy of target identification through complementary advantages. LiP-MS technology leverages conformational stability changes induced by small molecule-protein binding, using non-restrictive enzyme digestion to screen for protease-resistant regions and thus identify potential targets. Meanwhile, ABPP technology utilizes designed small molecule probes to covalently label target proteins, enabling functional verification of candidate targets. This combined approach retains the advantages of LiP-MS while using ABPP probes to verify the reliability of target identification results, effectively overcoming the limitations of traditional single-technique approaches such as high false-positive rates and complex probe designs.

[0058] The LiP-MS technique for target identification involves: protein extraction; non-restriction enzyme digestion; protein denaturation; trypsin digestion; mass spectrometry analysis; and data analysis. Because the positive probe synthesized in this invention contains a specific diacylpropidine fragment, it must first be incubated under 365nm UV light for 30 min before restriction enzyme digestion to ensure binding between the probe and the protein.

[0059] All samples were divided into four groups, as shown in Table 3: blank group, negative control group, low-dose group, and high-dose group. The drug concentration in the high-dose group was 1 μM, and in the low-dose group it was 0.1 μM. Each experiment was repeated three times to reduce error.

[0060] Table 3 Sample Grouping

[0061] Based on the principle of LiP-MS technology for identifying small molecule targets, this invention aims to identify long peptides with increasing abundance and short peptides with decreasing abundance within the aforementioned peptide sequences. These short peptides may be the ones that bind to small molecules. Then, by matching these peptides to their corresponding proteins, the identification of small molecule compound targets is completed. Taking HSPA5 as an example: this invention requires integrating upregulated and downregulated peptides from the same protein sequence together, allowing for a direct visualization of the changes in different peptide sequences within the same protein. Figure 10 As shown.

[0062] The differential abundance peptide mapping clearly shows that in HSPA5, there are peptide sequences where the abundance of long sequences increases and the abundance of short sequences decreases within the same sequence; or peptide sequences where the abundance of adjacent sequences increases and decreases simultaneously.

[0063] According to the NCBI CCDS database, the complete amino acid sequence of HSPA5, CCDS6863.1, was found. Comparison with the data above confirmed that the protein is HSPA5, a member of the heat shock 70 family of proteins. Figure 11 As shown.

[0064] Proteomics detection methods: In peptide screening based on differential expression analysis, a fold change (FC) > 1.5 (upregulation threshold) or < 0.667 (downregulation threshold) was used as the screening criteria. Combined with the statistical significance level (P < 0.05, through t-test or other test methods), the number of peptides with significant upregulation and downregulation between groups was finally screened out and represented in the form of a volcano plot. The proteins corresponding to the top ten peptides with the most significant upregulation and downregulation were labeled.

[0065] Cluster analysis is used to divide objects in a dataset into several groups (clusters) to maximize the similarity of samples within the same cluster and the difference between samples in different clusters.

[0066] Gene Ontology (GO) is a standardized biological terminology system that annotates proteins through gene ontology. GO functional analysis is mainly divided into three categories: (1) Biological Process (BP); (2) Molecular Function (MF); and (3) Cellular Component (CC). This paper uses Blast2Go (https: / / www.blast2go.com / ) software for GO analysis.

[0067] The KEGG pathway has seven primary categories: Metabolism; Genetic Information Processing; Environmental Information Processing; Cellular Processes; Organismal Systems; Human Diseases; and Drug Development. This invention utilizes the Kyoto Encyclopedia of Genes and Genomes database to analyze differentially expressed peptides.

[0068] Scratch test: After transfection, discard the culture medium and wash once with warm PBS. Carefully streak the cells with a 10 μL pipette tip, then carefully rinse away the streaked cells with warm PBS. Add complete culture medium and continue incubation. Take photos at 0 h, 3 h, 6 h, 12 h, 24 h and 48 h.

[0069] Dynamic simulation: from Figure 12 As can be seen, the amino acids HSP90AB1, HSPA2, HSPA5, and HSPA8 are the binding sites for trimethoprim. Subsequent mutations at these amino acid sites can be used to demonstrate whether trimethoprim binds to these sites, thus affecting the protein's function. Furthermore, this invention calculated the binding energies of HSP90AB1 to TA to be -12.0 kcal / mol, HSPA2 to trimethoprim to be -9.6 kcal / mol, HSPA5 to be -10.1 kcal / mol, and HSPA8 to be -7.5 kcal / mol.

[0070] The invention will subsequently undergo pull-down verification experiments.

[0071] The positive probe obtained in (1) was reacted with biotin to obtain the positive probe biotin small molecule, and its high-resolution mass spectrometry data are as follows: Figure 13 As shown: HRESIMS (negative) m / z 1301.7242 [MH]+ (calcd for C67H101N10O14S, 1301.7219).

[0072] ; Plasmids containing HSPA2, HSPA5, HSPA8, and HSP90AB1 were transfected for 24 h. Western blot analysis was then performed; after obtaining the whole protein, the positive probe biotin was added to the whole protein, and the mixture was incubated at 4°C for 6 h. Immunoblot bands are shown below. Figure 14 As shown in the figure, after overexpressing HSP family-related proteins, it was found that the probe of trienemycin could pull them down, indicating that trienemycin can bind to HSPs.

[0073] Following the pull-down validation experiment, this invention further validates the binding of triamcinolone acetonide to heat shock proteins using a cellular thermal displacement assay. The specific experiments are as follows: Combination Figure 15 Cell thermal displacement experiments using two pancreatic cancer cell lines demonstrated that trienemycin can indeed enhance the protein stability of HSPA2, HSPA5, and HSP90AB1, indicating that trienemycin can indeed bind to these proteins, and that the effects of trienemycin on different proteins differ. Further analysis of the data revealed that HSPA2 and HSP90AB1 showed greater variation at temperatures above 55℃, while HSPA5 showed relatively smaller variation within the same temperature range, suggesting that trienemycin has a stronger effect on HSPA5.

[0074] After verifying the binding of trienemycin and heat shock protein, this invention further verified the binding at the transcriptional and protein levels.

[0075] The changes in HSPA2, HSPA5, HSPA8, and HSP90AB1 at the transcriptional level were investigated using qPCR. The specific results are shown below: from Figure 16 It can be seen that the transcriptional level of HSP90AB1 increased, but was not significant in either cell line; the transcriptional level of HSPA2 increased, and was significant in both cell lines; the transcriptional level of HSPA5 increased, but was not significant in the PANC-1 cell line; the transcriptional level of HSPA8 decreased, but was not significant in MIA PaCa-2.

[0076] When studying changes in protein expression levels of related proteins, the drug was administered at approximately 60% cell density for 24 hours. Six concentration gradients were used: blank, 0.1 μM, 0.2 μM, 0.4 μM, and 0.8 μM. The obtained protein expression level results are as follows: Figure 17 As shown: Analysis of the protein expression levels of heat shock family proteins showed that in the PANC-1 cell line, the protein expression level of HSPA2 decreased by 50% at a drug concentration of 0.8 μM; while the protein expression level of HSPA5 decreased by 45% at a drug concentration of 0.1 μM; and the protein expression level of HSP90AB1 decreased by 25% at a drug concentration of 0.8 μM.

[0077] In the MIA PaCa-2 cell line, HSPA2 protein expression decreased by 30% at a drug concentration of 0.8 μM; HSPA5 protein expression decreased by 40% at a drug concentration of 0.1 μM; and HSP90AB1 protein expression decreased by 50% at a drug concentration of 0.8 μM. These data indicate that the addition of trimethoprim reduced the expression levels of the corresponding proteins in both pancreatic cancer cell lines.

[0078] The preceding analysis revealed an increase in the transcriptional levels of three heat shock proteins, suggesting that the decrease in their protein expression levels might be due to protein degradation. To verify this hypothesis, this invention first used the protein synthesis inhibitor CHX to inhibit the synthesis of heat shock family proteins, and then added trimethoprim to investigate whether the protein levels still decreased. The experiment was conducted in two groups: one group received only CHX; the other group received a mixture of CHX and trimethoprim to study the effects of time gradients on the protein expression levels of heat shock proteins.

[0079] Combination Figure 18 Data showed that protein synthesis was terminated after the addition of a protein synthesis inhibitor. The group receiving CHX + trimethoprim-treated combination showed a decrease in heat shock protein expression compared to the group receiving only CHX, indicating that the protein was degraded after treatment. The main degradation pathways of proteins are proteasome degradation and lysosomal degradation. Based on the above analysis, four experimental groups were first established to investigate whether proteins were degraded via the proteasome pathway: (1) blank; (2) trimethoprim-treated group; (3) MG132-treated group; (4) MG132 + trimethoprim-treated group, where MG132 is a proteasome inhibitor. Specific experimental results are as follows: Figure 19 As shown: The results indicate that even in the presence of MG132, trienemycin can still lead to a decrease in the protein expression levels of HSPA2 and HSPA5, suggesting that in addition to the proteasome pathway, trienemycin may also degrade proteins through other pathways, such as the lysosome pathway. The induction results of HSP90AB1, HSPA2, and HSPA5 in the presence of MG132 showed differences, mainly in that trienemycin did not significantly induce a decrease in the protein expression level of HSP90AB1, indicating that trienemycin primarily induces the degradation of HSP90AB1 through the proteasome pathway.

[0080] In addition to studying molecular mechanisms, this invention also investigates some phenotypic mechanisms. The scratch assay is a classic experimental protocol widely used in cell biology research. This invention investigates the effects of HSPA5 overexpression on cell invasion and migration in two pancreatic cancer cell lines.

[0081] Combination Figure 20 Based on the scratch assay results of the PANC-1 and MIA PaCa-2 cell lines, it was found that the scratches in the myc-HSPA5 group were thinner than those in the EV group, indicating that overexpression of HSPA5 promoted cancer cell migration; the scratches in the trienemycin group were thicker than those in the EV group, indicating that trienemycin inhibited cancer cell migration; the scratches in the myc-HSPA5+trienemycin group were thicker than those in the myc-HSPA5 group, indicating that even with overexpression of HSPA5, trienemycin could still inhibit cell migration.

[0082] Previous research showed that HSPA5 protein expression levels decreased with the addition of trimethoprim. This indicates that in both pancreatic cancer cell lines, trimethoprim inhibited cancer cell migration by suppressing HSPA5 protein expression.

[0083] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0085] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A trienemycin photoaffinity probe, characterized in that, include: Using Trienomycinol as a substrate, a substitution reaction was carried out with iodinated bisacrylidine, followed by deprotection of the propylidene to obtain the corresponding photoaffinity-negative probe; The corresponding photoaffinity positive probe was obtained by selectively esterifying Fmoc-D-alanine with the hydroxyl group at position 11 of the negative probe, removing the Fmoc protecting group on the nitrogen atom, and then condensing it with cyclohexane.

2. The trienycin photoaffinity-negative probe according to claim 1, characterized in that, The preparation of the aforementioned photoaffinity-negative probe specifically includes: Trienomycinol was dissolved in acetone and stirred evenly with 2,2-dimethoxypropane. At room temperature, a catalytic amount of racemic camphor sulfonic acid was added and stirred evenly. After the reactants were completely reacted, triethylamine was added and stirred at room temperature. The resulting liquid was evaporated under reduced pressure to obtain a crude product, which was then extracted with an organic solvent and dried to obtain compound 1. The molar ratio of Trienomycinol to racemic camphor sulfonic acid was 1:0.

01. Compound 1 and potassium carbonate were reacted with N,N-dimethylformamide and acetone at room temperature under an argon atmosphere and stirred. Then 3-(3-yne-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was added and stirred until homogeneous. The mixture was then sealed in the dark and reacted at 40 °C for 72 h. After extraction with an organic solvent and drying, compound 2 was obtained. The molar ratio of compound 1:potassium carbonate:3-(3-yne-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was 1:5:

5. After compound 2 was dissolved in methanol, a catalytic amount of racemic camphor sulfonic acid was added, and the mixture was sealed and stirred for about 1.5 h after being protected from light by paper. After the reaction of the raw materials was complete, triethylamine was added and stirred at room temperature for 30 min. On a molar ratio basis, compound 2: racemic camphor sulfonic acid = 1:0.

01.

3. The trienzyme photoaffinity positive probe according to claim 1 or 2, characterized in that, Selective esterification of the negative probe with Fmoc-D-alanine at the 11-hydroxyl position, followed by removal of the Fmoc protecting group on the nitrogen atom and condensation with cyclohexanecarboxylic acid, yields the corresponding photoaffinity positive probe, specifically including: Fmoc-D-alanine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalytic amount of 4-dimethylpyridine were added to dichloromethane and stirred until homogeneous at 0 °C under an argon atmosphere. After adding N,N-diisopropylethylamine and stirring until homogeneous, a negative probe was added. The mixture was then sealed and stirred in the dark to obtain compound 3. Molar ratio meter, photoaffinity-negative probe: Fmoc-D-alanine: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:5; Compound 3 was added to N,N-dimethylformamide at -40°C under an argon atmosphere and stirred until homogeneous; then a tetrahydrofuran solution of tetrabutylammonium fluoride was slowly added. After the addition of the compound was completed, the reaction was carried out in the dark to obtain compound 4; the molar ratio of compound 3 to tetrabutylammonium fluoride was 1:

5. Cyclohexane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and a catalytic amount of 4-dimethylpyridine were added to dichloromethane and stirred until homogeneous at -20 °C under an argon atmosphere; N,N-diisopropylethylamine was added and stirred until homogeneous, and then compound 4 from the previous step was added, and the reaction was carried out in the dark. On a molar ratio basis, compound 4: cyclohexanecarboxylic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 1-hydroxybenzotriazole: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:

5.

4. The trienycin photoaffinity probe according to claim 1 or 2, characterized in that, The Trienomycinol was obtained from the fermentation product of strain H2S5 by column chromatography, lithium aluminum hydride reduction and gel purification to obtain an intermediate Trienomycinol with a purity of over 80%.

5. The trienemycin photoaffinity probe according to claim 4, characterized in that, The specific process includes: Enrichment of secondary metabolites of strain H2S5: The H2S5 strain was inoculated into SPY medium and cultured in a constant temperature shaker at 27℃ and 120 rpm for 7 days. After the culture was completed, the bacterial cells and bacterial solution were separated. The bacterial solution was extracted three times with 50 L of ethyl acetate, and the organic phase was concentrated to finally obtain the extract. Separation of Trienomycinol: The extract was mixed with normal silica gel and eluted with ethyl acetate and dichloromethane:methanol = 10:1 as the eluent. The blue portion of the vanillin color reagent was collected and the eluent was concentrated. After drying the concentrate, it was reacted with lithium aluminum hydride in tetrahydrofuran in an anhydrous and oxygen-free system. After the reaction of the raw materials was complete, the reaction was quenched at 0°C with a saturated potassium sodium tartrate solution and stirred overnight at room temperature. Then, it was extracted three times with ethyl acetate, the organic phases were combined, washed and dried, and the resulting liquid was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography under gradient elution conditions from dichloromethane:methanol = 50:1 to dichloromethane:methanol = 10:1 by volume to obtain crude Trienomycinol. The crude Trienomycinol was purified by methanol gel column chromatography.

6. The method for preparing the trienemycin photoaffinity probe according to any one of claims 1-5, characterized in that, include: Using Trienomycinol as a substrate, a substitution reaction was carried out with iodopropyl diacylpyridine, followed by depropionation to obtain the corresponding photoaffinity-negative probe. Specifically, Trienomycinol was dissolved in acetone and stirred with 2,2-dimethoxypropane until homogeneous. At room temperature, a catalytic amount of racemic camphorsulfonic acid was added and stirred until homogeneous. After the reactants were completely reacted, triethylamine was added and stirred at room temperature. The resulting liquid was evaporated under reduced pressure to obtain a crude product, which was then extracted with an organic solvent and dried to obtain compound 1. The molar ratio of Trienomycinol to racemic camphorsulfonic acid was 1:0.

01. Compound 1 and potassium carbonate were added to N,N-dimethylformamide and acetone at room temperature under an argon atmosphere and stirred. Subsequently, 3-(3-ynyl-1-butyl)-3-(2-iodoethyl)-3H-bisacylpyridine was added and stirred until homogeneous. The mixture was then sealed in a light-proof environment and reacted continuously at 40 °C for 72 days. h; Compound 2 was obtained after extraction and drying with organic solvent; the molar ratio of compound 1: potassium carbonate: 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine was 1:5:5; after compound 2 was dissolved in methanol, a catalytic amount of racemic camphor sulfonic acid was added, and the mixture was sealed and stirred for about 1.5 h after being protected from light by paper; after the reaction of the raw materials was complete, triethylamine was added and stirred at room temperature for 30 min; the molar ratio of compound 2: racemic camphor sulfonic acid was 1:0.01; Selective esterification of Fmoc-D-alanine with the 11-hydroxyl group of the negative probe, followed by removal of the nitrogen-containing Fmoc protecting group and condensation with cyclohexanecarboxylic acid, yields the corresponding photoaffinity positive probe. Specifically, the probe consists of Fmoc-D-alanine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalytic amount of 4-dimethylpyridine, at 0... At -40°C under an argon atmosphere, dichloromethane was added and stirred until homogeneous. N,N-diisopropylethylamine was added and stirred until homogeneous, followed by the addition of a negative probe. After light protection, the reaction was sealed and stirred to obtain compound 3. The molar ratio of photoaffinity negative probe:Fmoc-D-alanine:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride:4-dimethylpyridine:N,N-diisopropylethylamine was 1:2:2:0.01:

5. Compound 3 was then reacted with N,N-dimethylformamide at -40°C under an argon atmosphere. The mixture was stirred until homogeneous. Then, a tetrahydrofuran solution of tetrabutylammonium fluoride was slowly added. After the addition was complete, the reaction was performed in the dark to obtain compound 4. The molar ratio of compound 3:tetrabutylammonium fluoride was 1:

5. Cyclohexane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and a catalytic amount of 4-dimethylpyridine were reacted at -20°C. At ℃, under an argon atmosphere, dichloromethane was added and stirred until homogeneous; N,N-diisopropylethylamine was added and stirred until homogeneous, then compound 4 from the previous step was added, and the reaction was carried out in the dark; on a molar ratio, compound 4: cyclohexanecarboxylic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 1-hydroxybenzotriazole: 4-dimethylpyridine: N,N-diisopropylethylamine = 1:2:2:0.01:

5.

7. The application of the trienemycin photoaffinity probe according to any one of claims 1-5 for the identification of trienemycin targets.

8. The application according to claim 7, wherein the target of trienemycin is identified by limited enzyme digestion mass spectrometry, and the target range is further narrowed by network pharmacology.

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