Use of tussilagone in the preparation of brd4 function inhibitors

Abstract

The present application relates to the technical fields of biological medicine, natural medicine and molecular target regulation, and particularly relates to application of faradiol in preparation of BRD4 function inhibitor. Through multi-level experiments such as calculation prediction, photoaffinity chemical proteomics, purified protein labeling, BLI, cell function, DNA damage and genetic intervention, the present application establishes the correlation between faradiol-BRD4-BD1-lung adenocarcinoma DNA damage.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, natural medicine and molecular targeted regulation technology, specifically to the application of coltsfoot ketone in the preparation of BRD4 functional inhibitors. Background Technology

[0002] Bromodomain-containing protein 4 (BRD4) is an important member of the bromodomain and superterminal domain (BET) family. It participates in the recruitment of transcription complexes, RNA polymerase II-related transcriptional regulation, cell cycle progression, and maintenance of DNA replication homeostasis by specifically recognizing histone acetylated lysine residues through its bromodomain. Studies have shown that aberrant activation of BRD4 is closely related to the proliferation, survival, and treatment resistance of various tumor cells. Therefore, BRD4 has become one of the important targets for anti-tumor drug development.

[0003] However, most existing BRD4 inhibitors are synthetic small molecules, such as JQ-1, OTX015, and ZEN-3694, with relatively concentrated chemical skeletons. Furthermore, there is still room for improvement in terms of selectivity, toxicity, pharmacokinetic properties, and applicable populations. In addition, some synthetic BRD4 inhibitors face problems such as drug resistance and adverse reactions in clinical applications. Therefore, developing naturally derived BRD4 inhibitors with different structural bases holds promise for providing new options for cancer treatment.

[0004] In the discovery of BRD4-targeting drugs, relying solely on molecular docking predictions or omics association analyses often fails to demonstrate the direct and specific interactions between candidate small molecules and the BRD4 protein, especially with specific bromine domains. Many candidate compounds, even those exhibiting antiproliferative activity at the cellular level, may still lack crucial evidence such as purified protein binding experiments, binding kinetic parameters, photoaffinity labeling, chemical proteomics identification, and target knockdown / knockout dependence verification, making it difficult to effectively distinguish between "direct targeting of BRD4" and "indirect influence on BRD4-related pathways via other proteins." Therefore, current technologies struggle to confirm the direct targeting effect of natural compounds on BRD4 and their target-dependent effects, which to some extent limits the application of natural products in BRD4-targeted therapy.

[0005] Tussilagone is a natural sesquiterpene compound isolated from the traditional Chinese medicine Tussilago farfara L., and has been reported to possess certain anti-inflammatory and antitumor biological activities. However, whether tussilagone can directly target BRD4, its direct interaction with the BRD4 domain, and the resulting biological effects have not yet been reported.

[0006] In summary, there is an urgent need in this field for a naturally derived compound that can directly target BRD4, induce DNA damage in cancer cells, and inhibit DNA synthesis, in order to overcome the shortcomings of existing synthetic BRD4 inhibitors in terms of selectivity, toxicity, and drug resistance, and to provide a new strategy for cancer treatment. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide the application of coltsfoot ketone in the preparation of BRD4 functional inhibitors.

[0008] The coltsone described in this invention has the molecular formula: C 23 H 34 O5, with a molecular weight of 390.51, has the following structural formula: .

[0009] This invention provides the application of coltsfoot ketone in the preparation of BRD4 functional inhibitors.

[0010] In some embodiments, the coltsone acts on the BD1 domain of BRD4.

[0011] This invention also provides the application of coltsone in the preparation of inhibitors acting on the BRD4-BD1 domain.

[0012] This invention also provides the application of coltsone in the preparation of BRD4-related functional modulatory agents.

[0013] In some embodiments, the direct interaction between coltsone and BRD4 can be verified by one or more of the following methods: photoaffinity probe labeling, parent competition experiments, purified protein labeling, biolayer interference (BLI), molecular docking, or molecular dynamics simulation.

[0014] In some embodiments, the experimental conditions for labeling the photoaffinity probe include: a probe concentration of 50 μM, an ultraviolet crosslinking wavelength of 365 nm, and a crosslinking time of 30 min.

[0015] In some embodiments, the conditions for the parent competition experiment include: the competition factor of free coltsoni is 10 times the probe concentration.

[0016] In some embodiments, the concentration range measured by the BLI includes 3.125 μM, 6.25 μM, 12.5 μM, or 25 μM.

[0017] In some embodiments, the effects of the BRD4 function inhibitor include at least one of the following: It can reduce BRD4-related transcriptional regulatory activity, interfere with BRD4-mediated protein complex or chromatin function, induce BRD4-dependent DNA damage, inhibit DNA synthesis, arrest the cell cycle, or promote tumor cell apoptosis.

[0018] In some embodiments, the induction of BRD4-dependent DNA damage includes at least one of the following: Increased γ-H2AX protein, increased γ-H2AX nuclear focal point, increased comet tail length, increased tail DNA percentage, increased tail moment, or changes in DNA damage response-related proteins.

[0019] In some embodiments, the working concentration of coltsone is 10-80 μM. For example, the working concentration of coltsone is 10, 12, 15, 18, 20, 23, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 63, 65, 68, 70, 72, 75, 78 or 80 μM.

[0020] In some embodiments, the working concentration of coltsone is 10-60 μM. For example, the working concentration of coltsone is 10, 12, 15, 18, 20, 23, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58 or 60 μM.

[0021] In some specific embodiments, the working concentration of coltsfoot ketone is 10, 50, or 80 μM.

[0022] In some embodiments, the treatment time for the BRD4 functional inhibitor is 36–60 hours. For example, the treatment time for the BRD4 functional inhibitor is 36, 40, 42, 45, 48, 50, 52, 55, 58, or 60 hours. Specifically, the treatment time for the BRD4 functional inhibitor is 48 hours.

[0023] Furthermore, the present invention provides a BRD4 functional inhibitor comprising coltsanthin and pharmaceutically acceptable excipients.

[0024] In some embodiments, the mass fraction of coltsone is 0.0001% to 10%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 3%, 5%, 8%, or 10%.

[0025] In some embodiments, the mass fraction of coltsone is 0.0001% to 5%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 3%, or 5%.

[0026] In some embodiments, the mass fraction of coltsone is 0.0001% to 1%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 0.3%, 0.5%, 0.8%, or 1%.

[0027] In some embodiments, the mass fraction of coltsfoot ketone is 0.0001% to 0.01%. For example, the mass fraction of coltsfoot ketone is 0.0001%, 0.000389%, 0.0005%, 0.0008%, 0.001%, 0.001943%, 0.003109%, 0.005%, 0.008%, or 0.01%.

[0028] In some specific embodiments, the mass fraction of coltsone is 0.000389%, 0.001943%, or 0.003109%.

[0029] In some embodiments, the mass concentration of coltsone is 1 to 100 μg / mL. For example, the mass concentration of coltsone can be 1, 3.905, 5, 8, 10, 15, 19.526, 31.241, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 μg / mL.

[0030] In some embodiments, the mass concentration of coltsone is 1 to 50 μg / mL. For example, the mass concentration of coltsone can be 1, 3.905, 5, 8, 10, 15, 19.526, 31.241, 35, 40, 45 or 50 to 100 μg / mL.

[0031] In some specific embodiments, the mass concentration of coltsone is 3.905 μg / mL, 19.526 μg / mL, or 31.241 μg / mL.

[0032] As a feasibility example, the coltsone is dissolved in a culture medium, with the culture medium density approximately 1 g / mL; When the concentration of coltsone is 10 μM, its mass concentration is 3.905 μg / mL, and its mass fraction is 0.000389%. When the concentration of coltsone was 50 μM, its mass concentration was 19.526 μg / mL, and its mass fraction was 0.001943%. When the concentration of coltsone is 80 μM, its mass concentration is 31.241 μg / mL and its mass fraction is 0.003109%.

[0033] In some embodiments, the excipients include at least one of a carrier, excipient, stabilizer, cosolvent, emulsifier, thickener, pH adjuster, antioxidant, preservative, buffer, osmotic pressure regulator, sustained-release material, or targeted delivery material.

[0034] In some embodiments, the carrier includes at least one of liposomes, nanoparticles, microspheres, microcapsules, micelles, emulsions, or dendritic macromolecules; The excipients include at least one of lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, starch, dicalcium phosphate, or calcium sulfate. The stabilizer includes at least one of polyethylene glycol, Tween, poloxamer, cyclodextrin, albumin, gelatin, or trehalose. The cosolvent includes at least one of polysorbate, polyethylene glycol, propylene glycol, glycerin, ethanol, poloxamer, or cyclodextrin; The pH adjuster includes at least one of citric acid, sodium citrate, phosphoric acid, phosphate, acetic acid, acetate, hydrochloric acid, sodium hydroxide, or tris(hydroxymethyl)aminomethane. The antioxidants include at least one of vitamin E, vitamin C, sodium sulfite, sodium metabisulfite, sodium thiosulfate, cysteine, or methionine. The preservative includes at least one of benzalkonium chloride, benzyl alcohol, p-hydroxybenzoate, thimerosal, or trichlorotert-butanol. The buffer includes at least one of phosphate buffer, citrate buffer, acetate buffer, borate buffer, or histidine buffer; The osmotic pressure regulator includes at least one of sodium chloride, glucose, mannitol, sorbitol, or glycerol; The sustained-release material includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, chitosan, sodium alginate, gelatin, hyaluronic acid, or hydroxypropyl methylcellulose. The targeted delivery material includes at least one of folic acid, transferrin, RGD peptide, hyaluronic acid, antibody or fragment thereof, aptamer or nucleic acid aptamer.

[0035] In some embodiments, the dosage form of the BRD4 functional inhibitor includes at least one of the following: injection, lyophilized powder for injection, liposome, nanoformulation, microsphere formulation, or oral formulation.

[0036] Furthermore, the present invention also provides the use of coltsfoot ketone in the preparation of medicaments for the prevention and / or treatment of diseases related to BRD4 overexpression.

[0037] In some embodiments, the drug targets BRD4.

[0038] In some specific embodiments, the drug targets the BD1 domain of BRD4.

[0039] In some embodiments, the prevention and / or treatment includes at least one of the following: increasing γ-H2AX, increasing comet tail moment, reducing cell viability, inhibiting cell proliferation, inhibiting cell migration, inhibiting DNA synthesis, promoting apoptosis, or causing DNA damage.

[0040] In some embodiments, the BRD4 overexpression-related diseases include non-small cell lung cancer and small cell lung cancer; wherein non-small cell lung cancer includes at least one of lung adenocarcinoma, lung squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, and sarcomatoid carcinoma; and the small cell lung cancer includes at least one of small cell carcinoma, mixed small cell carcinoma, or small cell neuroendocrine carcinoma.

[0041] In some specific embodiments, the BRD4 overexpression-related disease is lung adenocarcinoma.

[0042] In some embodiments, the lung adenocarcinoma includes at least one of A549, Calu-3, PC9, HCC827, and H1975; The lung squamous cell carcinoma includes at least one of H520, SK-MES-1, and NCI-H1703; The large cell carcinoma includes at least one of H460, H1299, and H661; The small cell lung cancer includes at least one of H69, H82, H446, and SHP-77.

[0043] Furthermore, the present invention also provides medicaments for the prevention and / or treatment of diseases related to BRD4 overexpression, including coltsanthin and pharmaceutically acceptable excipients.

[0044] In some embodiments, the mass fraction of coltsone is 0.0001% to 10%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 3%, 5%, 8%, or 10%.

[0045] In some embodiments, the mass fraction of coltsone is 0.0001% to 5%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 3%, or 5%.

[0046] In some embodiments, the mass fraction of coltsone is 0.0001% to 1%. For example, the mass fraction of coltsone is 0.0001%, 0.001%, 0.01%, 0.1%, 0.3%, 0.5%, 0.8%, or 1%.

[0047] In some embodiments, the mass fraction of coltsfoot ketone is 0.0001% to 0.01%. For example, the mass fraction of coltsfoot ketone is 0.0001%, 0.000389%, 0.0005%, 0.0008%, 0.001%, 0.001943%, 0.003109%, 0.005%, 0.008%, or 0.01%.

[0048] In some specific embodiments, the mass fraction of coltsone is 0.000389%, 0.001943%, or 0.003109%.

[0049] In some embodiments, the excipients include at least one of a carrier, excipient, stabilizer, cosolvent, emulsifier, thickener, pH adjuster, antioxidant, preservative, buffer, osmotic pressure regulator, sustained-release material, or targeted delivery material.

[0050] In some embodiments, the carrier includes at least one of liposomes, nanoparticles, microspheres, microcapsules, micelles, emulsions, or dendritic macromolecules; The excipients include at least one of lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, starch, dicalcium phosphate, or calcium sulfate. The stabilizer includes at least one of polyethylene glycol, Tween, poloxamer, cyclodextrin, albumin, gelatin, or trehalose. The cosolvent includes at least one of polysorbate, polyethylene glycol, propylene glycol, glycerin, ethanol, poloxamer, or cyclodextrin; The emulsifier includes at least one of lecithin, soybean lecithin, cholesterol, Tween, Span, or poloxamer. The thickener includes at least one of hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, carbomer, sodium alginate, polyvinylpyrrolidone, or polyethylene glycol; The pH adjuster includes at least one of citric acid, sodium citrate, phosphoric acid, phosphate, acetic acid, acetate, hydrochloric acid, sodium hydroxide, or tris(hydroxymethyl)aminomethane. The antioxidants include at least one of vitamin E, vitamin C, sodium sulfite, sodium metabisulfite, sodium thiosulfate, cysteine, or methionine. The preservative includes at least one of benzalkonium chloride, benzyl alcohol, p-hydroxybenzoate, thimerosal, or trichlorotert-butanol. The buffer includes at least one of phosphate buffer, citrate buffer, acetate buffer, borate buffer, or histidine buffer; The osmotic pressure regulator includes at least one of sodium chloride, glucose, mannitol, sorbitol, or glycerol; The sustained-release material includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, chitosan, sodium alginate, gelatin, hyaluronic acid, or hydroxypropyl methylcellulose. The targeted delivery material includes at least one of folic acid, transferrin, RGD peptide, hyaluronic acid, antibody or fragment thereof, aptamer or nucleic acid aptamer.

[0051] In some embodiments, the dosage form of the drug includes at least one of injection, lyophilized powder for injection, liposome, nanoformulation, microsphere formulation, or oral formulation.

[0052] In some embodiments, the oral formulation includes at least one of tablets, capsules, granules, powders, pills, oral liquids, suspensions, emulsions, syrups, or drops; The nanoformulation includes at least one of nanoparticles, nanoemulsions, nanosuspensions, polymer micelles, lipid nanoparticles, or nanocrystals. The injectable includes at least one of the following: solution-type injectable, suspension-type injectable, emulsion-type injectable, sterile powder for injection, or concentrated solution for injection.

[0053] The beneficial effects of this invention are as follows: (1) This provides new uses for coltsone as a BRD4 function inhibitor and a BRD4-BD1 action compound.

[0054] (2) A direct target evidence chain consisting of computational prediction, photoaffinity chemical proteomics, purified protein labeling and BLI was established.

[0055] (3) The technical effects of inhibiting cell viability, migration, colony formation and DNA synthesis and promoting apoptosis were verified in A549 and Calu-3.

[0056] (4) Reproducible evidence of DNA damage was obtained through γ-H2AX and comet experiments.

[0057] (5) Provide BRD4 dependence support by simulating BRD4 knockdown and the weakening of drug efficacy after target depletion.

[0058] (6) Coltsfoot ketone can be further used as an active core for formulation development, structural optimization or targeted delivery systems.

[0059] This invention relates to the application of coltsfoot ketone in the preparation of BRD4 functional inhibitors. Potential target prediction and molecular docking suggest that coltsfoot ketone can stably bind to the BD1 domain of BRD4. Coltsfoot ketone photoaffinity probes containing a diazidopropylidine photocrosslinking group and a terminal alkyne group were used to identify BRD4 via UV crosslinking, click chemistry, affinity enrichment, and mass spectrometry. The absence of probes, photocrosslinking, and competition from excess free coltsfoot ketone reduced BRD4 enrichment. Purified BRD4 protein labeling and biolayer interference further demonstrated the direct interaction between coltsfoot ketone and BRD4. Coltsfoot ketone can reduce the viability, migration, colony formation, and DNA synthesis of A549 and Calu-3 lung adenocarcinoma cells, promote apoptosis, and increase γ-H2AX and comet tail moments. Furthermore, Si BRD4 and Sh BRD4 weaken the newly induced apoptosis effect of coltsfoot ketone, suggesting that the anti-lung adenocarcinoma effect of coltsfoot ketone is at least partially dependent on BRD4. This invention provides a new technical solution for the development of coltsfoot ketone as a BRD4 functional inhibitor and a candidate drug for anti-lung adenocarcinoma. Attached Figure Description

[0060] Figure 1 The effect of 28 Chinese herbal monomers on total BRD4 protein was detected by Western blotting (reference table 1 for 28 Chinese herbal monomers). Figure 2 The graph shows the inhibitory effects of 28 Chinese herbal medicine monomers on tumor cell proliferation and the effect of candidate drugs on total BRD4 protein (A is the tumor inhibitory effect of 28 Chinese herbal medicine monomers detected by CCK8, B is the detection result of the relative protein expression level of BRD4 (BRD4 / GAPDH) in A549 cells after treatment with 28 Chinese herbal medicine monomers, and C is the effect of candidate drugs on total BRD4 protein). Figure 3 Proteomic identification results for three candidate drugs; Figure 4 SwissTargetPrediction identifies BRD4 as a potential target for coltsfoot ketone (A shows the SwissTargetPrediction analysis workflow; B shows the top 50 predicted candidate targets and their category distribution; C shows the top 30 signaling pathways enriched by KEGG in coltsfoot transcriptome sequencing; D shows the signaling pathways enriched by KEGG in coltsfoot proteome sequencing; E shows the 12 signaling pathways found at the intersection of KEGG enrichment from coltsfoot transcriptome sequencing and proteome sequencing; F shows the predicted target and E...). Figure 12 PPI network analysis of KEGG pathways). Figure 5 The docking conformation and molecular dynamics stability of coltsone with BRD4-BD1 are shown in Figure 1. (A. Overall binding position of coltsone in BRD4-BD1, Vina score = -7.8 kcal / mol; B. 3D magnified view of the coltsone binding pocket; C. 2D interaction diagram of coltsone and BRD4-BD1). Figure 6 The design, quality control, and activity verification of the coltsone photoaffinity probe were carried out (Figure A shows the synthesis steps of the coltsone-bisacrididine alkyne probe; Figure B shows the probe workflow; Figure C shows the Wester blot bands of four samples after copper click chemical linking to biotin, competing with DMSO, uncrosslinked, probe, and 10-fold coltsone, demonstrating the effectiveness of the coltsone-bisacrididine alkyne probe). Figure 7 BRD4 was identified using ABPP / photoaffinity proteomics (Figure A shows the overall labeling of probe-UV, probe+UV, and parent competing group; Figure B shows the enrichment of BRD4 and representative peptides in mass spectrometry). Figure 8 The purified BRD4-BD1 protein was directly labeled with a coltsone probe (A is the protein immunoblotting of purified BRD4 protein after it was chemically linked to biotin by a copper-click probe in four competitive groups: no probe, probe-UV, probe+UV, and probe+excess free coltsone; B is the gray-scale quantification of the SA-HRP band signal in A, which was normalized to 1 with the DMSO group, and the result is expressed as the fold change relative to the DMSO group). Figure 9 To confirm the direct binding of talcone to BRD4 using biolayer interferometry (BLI), Figure A shows the binding and dissociation processes of different concentrations of talcone (3.125, 6.25, 12.5, and 25 μM) with BRD4, detected by BLI; KD = 1.3 ± 0.09 μM, with a good fit R² = 0.9873; Figure B summarizes the BLI kinetic fitting parameters: kon = 1.9 ± 0.10 × 10⁻⁶. 4 M -1 S -1 koff = 2.17 ± 0.05 × 10 -2 S -1 (and KD=1.3 ± 0.09 μM). Figure 10 Coltstilbene inhibits the viability of A549 and Calu-3 cells; Figure 11 Coltsedge inhibits A549 cell viability in a BRD4-dependent manner; Figure 12 Coltsfoot ketone inhibits the migration and colony formation of lung adenocarcinoma cells (A: Coltsfoot ketone inhibits the migration of lung adenocarcinoma cells; B: Coltsfoot ketone inhibits the colony formation of lung adenocarcinoma cells). Figure 13 Coltsfoot ketone inhibits DNA synthesis and promotes apoptosis in lung adenocarcinoma cells (A: EDU detection of coltsfoot ketone inhibiting DNA synthesis in lung adenocarcinoma cells; B: Annexin V / PI double staining detection of coltsfoot ketone inducing apoptosis in lung adenocarcinoma cells). Figure 14 Coltsfoot ketone upregulates γ-H2AX and increases the focal points of nuclear DNA damage (A is the increase in γ-H2AX protein caused by coltsfoot ketone treatment as detected by Western blotting; B is the increase in γ-H2AX caused by coltsfoot ketone treatment as detected by immunofluorescence, and it is nuclear localized). Figure 15 To verify the DNA damage induced by coltsfoot ketone in lung adenocarcinoma cells using comet experiments; Figure 16 Knockdown of BRD4 weakens the new apoptosis effect induced by coltsfoot ketone. Detailed Implementation

[0061] This invention provides the application of coltsone in the preparation of BRD4 functional inhibitors. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0062] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0063] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0064] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0065] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0066] The terms “tussilagone”, “TSG”, and “TUS” are used interchangeably and all refer to natural sesquiterpenoid compounds derived from Tussilago farfara flowers; in this application, they refer to tussilagone and its pharmaceutically acceptable forms that meet experimental or pharmaceutical purity requirements.

[0067] The term "BRD4" refers to Bromodomain-containing protein 4, a member of the BET family, which is involved in chromatin recognition, transcriptional elongation, cell cycle, and DNA damage response.

[0068] The term "BD1" refers to the first bromine domain of BRD4. In this application, it is supported by ABPP, purified protein labeling, BLI, and computational simulations as an important binding region for coltsanthin.

[0069] The term "BRD4 function inhibitor" refers to compounds that can directly or indirectly reduce BRD4-mediated biological functions, including but not limited to reducing BRD4-related transcriptional elongation, DNA replication homeostasis, or tumor cell survival.

[0070] The term "ABPP" or "AfBPP" refers to a chemical proteomics / active proteomics method based on photoaffinity probes. The probe contains a diaziridine photocrosslinking group and a terminal alkyne group, and the target protein is identified by UV crosslinking, click chemistry, affinity enrichment, and mass spectrometry.

[0071] The term "BLI" refers to Bio-layer interferometry, a technique used to measure the binding and dissociation of small molecules with proteins in real time.

[0072] The term "γ-H2AX" refers to biomarkers associated with DNA double-strand breaks and replication stress.

[0073] The term "comet assay" refers to single-cell gel electrophoresis, used to quantitatively evaluate DNA breaks, with metrics including tail length, tail DNA percentage, and tail moment.

[0074] The term "lung adenocarcinoma" refers to the main pathological type of non-small cell lung cancer; the in vitro embodiments of this application include at least A549 and Calu-3 cells.

[0075] All experiments in this application were independently replicated biologically, with n≥3 replicates; data are expressed as mean±SD or mean±SEM; statistical analysis was performed using t-tests, and p<0.05 was considered statistically significant.

[0076] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0077] This invention establishes for the first time a multi-layered chain of evidence for the direct interaction between coltsone and the BRD4-BD1 domain, and further demonstrates that this interaction can regulate BRD4-related functions, producing detectable effects such as DNA damage, DNA synthesis inhibition, and apoptosis.

[0078] The experimental results are as follows: This application reveals that coltsanthin can enter lung adenocarcinoma cells and interact with BRD4. SwissTargetPrediction and molecular docking suggest that BRD4-BD1 is a potential binding region; coltsanthin diacylpropionyl-alkynyl photoaffinity probes, after UV cross-linking, can enrich BRD4 in the cytoproteome, and this enrichment is significantly affected by the competitive conditions of probe-free, UV-free, and excessive free coltsanthin; purified BRD4 protein can be directly labeled with the probe; BLI further confirms that coltsanthin has a measurable direct binding to BRD4.

[0079] In A549 and Calu-3 lung adenocarcinoma cells, coltsfoot reduces cell viability, colony formation, and migration, inhibits EdU incorporation, and promotes apoptosis. Coltsfoot treatment increases γ-H2AX expression and nuclear focal points, along with comet tail moments and the proportion of comet-positive cells, indicating that it can induce DNA damage. According to literature reports, these changes are highly correlated with the inhibition of BRD4-BD1 domain function. Sh BRD4 and Si BRD4 treatments decreased the sensitivity of A549 cells to coltsfoot, suggesting that the anti-lung adenocarcinoma effect of coltsfoot is at least partially dependent on BRD4.

[0080] Based on the above findings, this application defines coltsone as a natural small molecule candidate that can directly interact with BRD4 and regulate BRD4-related functions.

[0081] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0082] All the test materials used in this invention are common commercial products and can be purchased on the market. Among them, tussilagone was purchased from Chengdu Mansite Co., Ltd., product number A0956, with a purity of ≥98%; it was dissolved in DMSO before use, with a final concentration range of 10~60 μM.

[0083] Other drugs used in the examples (such as traditional Chinese medicine monomers) were purchased from Chengdu Mansite.

[0084] A549 and Calu-3 cell lines were purchased from the Shanghai Cell Resource Center (affiliated with the Chinese Academy of Sciences). The culture medium was Cytiva SV30303.01 DMEM (high glucose version).

[0085] BRD4 protein: full-length or domain constructs BRD4 E49-E460, purchased from MedChemExpress, catalog number HY-P7846; BD1 domain constructs (E49-E170), purchased from SinoBiological, catalog number B18-31H; all with a purity >90%.

[0086] BRD4 antibody was purchased from MedChemExpress, catalog number HY-P80976; γ-H2AX antibody was purchased from Zenbio, catalog number R381558; and GAPDH antibody was purchased from Proteintech, catalog number 60004-1-Ig.

[0087] The present invention will be further illustrated below with reference to the embodiments: Example 1: Screening of medicinal monomers Virtual screening was conducted on a database of 1000 traditional Chinese medicine monomers. Following a literature review, 28 monomers with reported anti-tumor effects were identified: typhain, demethylene berberine, yohimbine hydrochloride, sophoridine; macaine, sarstigmol, glycyrrhizic acid monoammonium salt; glycyrrhizic acid ammonium, morin C, tetrahydroberberine, rutin, kaempferol-3-O-rutin, 2,3,5,4'-tetrahydroxystilbene glucoside, rhein-8-O-glucoside, and montmorillonite. Anthocyanins, grandiol, apigenin-7-O-β-D-glucopyranoside; cosmosin, geraniol-7-O-β-D-glucopyranoside, β,β-dimethylacryloylshikonin; β,β-dimethylacryloylarcinin, emodin-8-β-D-glucopyranoside, physalisin, α-dextrin, rutin (HPLC grade), baicalin; scutellarin, echinacoside, cimicifugain, isorhamnetin-3-O-neohesperidin, stemona alkaloid, camptothecin, coltsfoot ketone (Table 1).

[0088] The effects of these 28 traditional Chinese medicine monomers on BRD4 total protein were detected by Western blotting, and their tumor-suppressive effects were detected by CCK8 assay. Combining the results of these two screening methods, three candidate drugs were selected: TSG, CPT, and DMAS. Figure 1 and Figure 2 Proteomic analysis of the three candidate drugs revealed that tsurulfone (TSG) had the most significant impact on BRD4 expression levels. Figure 3 After comparison, TSG showed a more significant effect on BRD4 expression levels and tumor suppression, therefore TSG was selected as the subject of subsequent research.

[0089] Table 1 28 kinds of Chinese medicinal monomers

[0090] Example 2: SwissTargetPrediction - Potential Target Prediction

[0091] Standardized SMILES or InChIKey data for coltsfoot ketone were input into the SwissTargetPrediction platform, with Homo sapiens selected as the species. The predicted target name, UniProt number, target category, and probability value were exported. Intersection or sequencing analyses were performed with the transcriptome and proteome-related gene sets after coltsfoot ketone application. KEGG pathway enrichment analysis of the transcriptome and proteome of A549 cells after coltsfoot ketone action revealed significant differences in multiple signaling pathways. Intersection of the KEGG enrichment data from transcriptome and proteome sequencing yielded 12 identical signaling pathways. These 12 pathways were then combined with the SwissTargetPrediction predicted targets for PPI network analysis.

[0092] Table 2. SwissTargetPrediction Target Prediction Results

[0093] The results showed that BRD4 was the 24th target predicted by SwissTargetPrediction (Table 2), and multiple KEGG pathways from transcriptome and proteome sequencing were associated with BRD4. Figure 4 PPI network analysis, combining the common pathways of transcriptome and proteome sequencing with SwissTargetPrediction predicted targets, revealed that BRD4 was the protein closest to the core. Subsequent ABPP mass spectrometry and direct binding results further confirmed BRD4 as the key validation target.

[0094] Example 3: Molecular docking and molecular dynamics simulation of colchicine with BRD4-BD1

[0095] The crystal structure of BRD4-BD1 was obtained from the Protein Data Bank (PDB ID: 0004lyi). The protein underwent dehydration, hydrogen supplementation, residue repair, and energy minimization. A docking grid was constructed using CB-dock2, with the pocket center coordinates set to (-12, -6, -2) and the box size to (23, 23, 23). Coltsfoot ketone was used for conformational generation and charge manipulation to achieve high-precision molecular docking, selecting a complex with the lowest binding energy and a reasonable conformation.

[0096] The results showed that coltsfoot ketone is highly likely to bind to the BD1 domain of the BRD4 protein. Figure 5 ).

[0097] Example 4: Design, Synthesis, and Activity Maintenance of the Aspen Photoaffinity Probe

[0098] Under nitrogen protection, p-toluenesulfonic acid monohydrate (1.0 equiv, 14 mg) was added to 2 mL of a methanol solution of coltsone (30 mg, 0.076 mmol, 1.0 equiv.), and the mixture was heated to 40°C and reacted for 12 hours. The reaction solution was concentrated and separated by thin-layer chromatography using petroleum ether solution containing 10% ethyl acetate as the developing solvent. A diazidopropylidine photocrosslinking group and a terminal alkyne group were introduced at sites that did not significantly disrupt the activity of the coltsone core to obtain the coltsone photoaffinity probe (hereinafter referred to as TSG-probe). The probe (50 μM) was incubated with live cells for 1 hour, thoroughly washed with PBS, and crosslinked under 365 nm UV light on ice for 30 minutes. Protein was lysed in 30 dishes of EDTA-free RIPA lysis buffer on ice, followed by BCA protein quantification and normalization. Biotin was chemically linked to copper-click lysis. Western blotting was then performed. The probe-labeled protein was detected using streptavidin-HRP. Setup: DMSO, photocrosslinking without UV irradiation, and 10 times free coltsfoot ketone were used as controls.

[0099] The results showed that TSG-Probe could effectively bind to live cell proteins and was competitively bound by free coltsoni ( Figure 6 ).

[0100] Example 5: ABPP / photoaffinity chemistry proteomics identification of BRD4

[0101] In the experimental group, live cells were incubated with the probe (50 μM) for 1 h, thoroughly washed with PBS, and cross-linked under 365 nm UV light on ice for 30 min. Proteins were then lysed on ice for 30 min using EDTA-free RIPA lysis buffer, followed by BCA protein quantification and normalization. Copper click chemistry was performed at 37°C for 30 min to ligate biotin. The sample ratio was methanol:chloroform:water = 1:4:1:3 to remove copper click chemistry impurities and free biotin. The mixture was incubated overnight with streptavidin magnetic beads. Differentially enriched proteins were identified by LC-MS / MS.

[0102] The results showed that BRD4 was detected as a specific peptide and significantly enriched in the probe + UV group; the BRD4 signal decreased under probe-free, UV-free, and excessive free coltsfoot ketone competitive conditions. These results support the labeling's probe dependence, light dependence, and parent competitiveness. Figure 7 ).

[0103] Example 6: Direct photocrosslinking labeling of purified BRD4-BD1 protein

[0104] The purified BRD4 protein was incubated with TUS-probe in PBS at 37°C for 1 h. After UV crosslinking on ice for 30 min, biotin was ligated via click chemilinking and detected by Western blotting. DMSO, probe-UV, and probe + excess free coltsone were used as control groups.

[0105] The results showed that the probe + UV group exhibited a clear BRD4 labeling signal; the signal was significantly weakened in the no-UV and no-probe groups; excessive free coltsfoot ketone competitively reduced probe labeling. This experiment ruled out indirect enrichment mediated by other intracellular proteins and directly supported the interaction between the coltsfoot ketone probe and the BRD4 protein. Figure 8 ).

[0106] Example 7 BLI determination of the binding kinetics between coltsanthin and BRD4-BD1

[0107] 50 μg of purified protein was taken from BRD4. Following the instructions of the biotinylation kit (Genemore), 0.25 μL of biotin was added. The mixture was incubated at room temperature for 45 min. The solution was then added to a desalted column equilibrated with PBS. After the entire column was filled, 400 μL of PBS was added. Once 400 μL of eluent was reached, another 400 μL of PBS was added, and the eluent was collected. Then, 200 μL of PBS was added, and the eluent was collected again. Two probes (Sartorius) were selected, and the baseline, loading, and baseline2 programs were executed sequentially to achieve baseline stabilization, antibody protein loading, and baseline stabilization after target protein loading. A double-subtraction experimental group was designed for the experiment. Two probes immobilized the target protein, and two probes did not immobilize the target protein. The probes with immobilized target protein and the probes without immobilized target protein were passed sequentially through the experimental group and the control group, and the baseline, association, and dissociation programs were executed. This achieved baseline stabilization, binding of the small molecule to the target protein, and dissociation of the small molecule from the target protein. The probes and the instrument recorded the signal changes throughout the process in real time. The error is doubled by subtracting the data from (immobilized target protein probe in the experimental group - unimmobilized target protein probe in the experimental group) and (immobilized target protein probe in the control group - unimmobilized target protein probe in the control group).

[0108] The results show that ( Figure 9 The KD of coltsone bound to purified BRD4 was 1.3 ± 0.09 μM, and the kon value was 1.9 ± 0.10 × 10⁻⁶ μM. 4 M -1 S -1 and koff = 2.17 ± 0.05 × 10-2 S -1 It has a relatively strong binding affinity to proteins among natural compounds (<10μM).

[0109] Example 8: Ascorbyl inhibits the viability of A549 and Calu-3 cells.

[0110] A549 and Calu-3 cells were seeded in 96-well plates and treated with different concentrations of coltsfoot ketone for 48 hours after 16-20 hours. CCK-8 culture medium was prepared at a ratio of 1:9 (CCK-8:complete medium). The original medium was discarded, and 100 μl of CCK-8-containing complete medium was added to each well. A blank control was included. The plates were incubated at 37°C for approximately 2 hours, and absorbance was measured at 450 nm using a microplate reader. The IC50 was calculated using nonlinear regression.

[0111] The results showed that coltsfoot ketone reduced the viability of two types of lung adenocarcinoma cells in a dose-dependent manner. The IC50 for A549 was 35.15 μM, and the IC50 for Calu-3 was 30.6 μM. Figure 10 ).

[0112] Example 9: Aspartate inhibits A549 cell viability-dependent BRD4

[0113] A549 cell lines were stably transfected to construct Sh NC, Ov BRD4, and Sh BRD4 cell lines, which were then seeded in 96-well plates. After 16–20 h, the cells were treated with different concentrations of coltsfoot ketone for 48 h. CCK-8 culture medium was prepared at a ratio of 1:9 (CCK-8:complete medium). The original medium was discarded, and 100 μl of CCK-8-containing complete medium was added to each well. A blank control was included. The plates were incubated at 37°C for approximately 2 h, and the absorbance was measured using a microplate reader at 450 nm. The IC50 was calculated using nonlinear regression.

[0114] The results showed that stable overexpression of BRD4 had no significant effect on the coltsone sensitivity of A549. However, at intermediate concentrations, BRD4 significantly reduced the coltsone sensitivity of A549. Figure 11 ).

[0115] Example 10: Parabens inhibit lung adenocarcinoma cell migration and colony formation.

[0116] Scratch assay: A549 and Calu-3 cells were seeded in 6-well plates. After reaching approximately 90% confluence, uniform-width scratches were created. Under low serum conditions, 15 μM coltsone was added, and images were taken at 0 h and 24 h.

[0117] Colony formation: Low-density cells were seeded in 6-well plates, treated with the appropriate concentration of coltsfoot ketone for 10 days, stained with crystal violet, and the number and area of ​​colonies were counted.

[0118] The results showed that tuftolone reduced scratch healing rate and significantly decreased clone number, supporting its ability to inhibit migration and long-term proliferation. Figure 12 ).

[0119] Example 11: Aspartate inhibits DNA synthesis and promotes apoptosis

[0120] EdU assay: A549 and Calu-3 cells were seeded in 96-well plates. After adhesion and reaching approximately 30% confluence, cells were treated with 0.30 μM coltsoni for 48 hours. The experiment was then performed according to the Beyotime EDU assay kit instructions. The old culture medium was discarded, and preheated EDU (37°C) was added. The cells were incubated for 2 hours, the supernatant was discarded, and the cells were thoroughly washed with PBS. Fixation was performed with 4% paraformaldehyde at room temperature for 15 minutes. The fixative was discarded, and the cells were thoroughly washed with PBS. Permeabilization was performed with 0.3% Tritium X-100 for 15 minutes, followed by thorough PBS washing. Click buffer was added, and the cells were incubated at room temperature in the dark for 30 minutes. The cells were thoroughly washed with PBS, and DAPI was added. The cells were incubated at room temperature in the dark for 10 minutes. Color development was then performed, and the EdU positivity rate was calculated.

[0121] Apoptosis assay: A549 and Calu-3 cells were seeded in 6 cm culture dishes and treated with different concentrations of coltsfoot ketone for 48 hours after 16-20 h. The proportions of early, late, and total apoptosis were detected by flow cytometry using Annexin V-FITC / PI double staining.

[0122] The results showed that coltsfoot ketone reduced the EdU positivity rate in A549 and Calu-3 cells and increased the apoptosis rate, indicating that it can inhibit DNA synthesis and promote cell death. Figure 13 ).

[0123] Example 12: The focus of aspen-induced γ-H2AX elevation and nuclear DNA damage.

[0124] A549 and Calu-3 cells were cultured in 6 cm dishes and treated with different concentrations of coltsfoot ketone for 48 hours after 16-20 h. Cells were collected, lysed on ice using RIPA for 30 min, and protein concentrations were standardized after BCA quantification. Western blotting was used to detect the expression level of γ-H2AX, with GAPDH as an internal control. Image J was used for grayscale statistical analysis of the γ-H2AX / GAPDH ratio, and normalization was performed with a coltsfoot ketone concentration of 0 as the baseline.

[0125] Immunofluorescence: A549 and Calu-3 cells were seeded in 96-well plates and treated with different concentrations of coltsfoot ketone for 48 hours after 16-20 hours. Cells were pre-cooled to -20°C with methanol, fixed at -20°C for 30 min, thoroughly washed with PBS, blocked with 3-5% BSA at room temperature for 1 h, and incubated overnight at 4°C with γ-H2AX antibody diluted with 3% BSA. Afterwards, cells were thoroughly washed with PBS, incubated with fluorescent secondary antibody at room temperature for 2 h, followed by DAPI incubation at room temperature for 10 min, and thoroughly washed with PBS. Images were acquired using uniform exposure parameters to quantify the average fluorescence intensity, the proportion of positive cells, or the number of focal points per nucleus.

[0126] The results showed that coltsfoot ketone increased γ-H2AX protein levels and nuclear focal signal, and this effect was dose- or time-dependent. Figure 14 ).

[0127] Example 13: Comet Experiment Verification of Coltsfoot-Induced DNA Breaks

[0128] Cells were collected 24 hours after treatment, resuspended in 100 μL PBS, and stored at 4°C. NMPA suspension was thawed at 90°C, incubated at 60°C, and the slide was preheated. 100 μL of gel was dropped onto the junction of the frosted and smooth surfaces of the slide, immediately covered with a coverslip, and the gel was spread out, avoiding air bubbles. The gel was cured in a humidified chamber at 4°C for 2 hours. LMPA suspension was thawed at 99°C, incubated at 90°C, and the slide was preheated at 40°C. After the lower gel layer solidified, the coverslip was carefully removed, and 20 μL of cell PBS suspension was mixed with 80 μL of 0.5% LMPA. 40 μL of cell gel suspension was dropped onto the lower agarose gel, covered with a coverslip, and the gel was spread out, avoiding air bubbles. The gel was cured in a humidified chamber at 4°C for 30 minutes. Lysis buffer was prepared and pre-cooled at 4°C. After the upper gel has solidified, remove the coverslip, slowly immerse the slide in the lysis buffer, and place it in a refrigerator at 4°C for lysis for 2 hours. Discard the lysis buffer, rinse with ultrapure water, 1 min × 3 times. Place the slide in a horizontal electrophoresis tank, immerse it in the electrophoresis buffer, and unwind in the dark for 20 min. Perform constant current electrophoresis at 250 mA for 20 min. Remove the slide, immerse it in the neutralization buffer, and neutralize for 5 min × 3 times. Absorb excess neutralization buffer with absorbent paper, add 50 μL of PI staining solution, and incubate at room temperature in the dark for 10 min. Wash away the staining solution with ultrapure water, cover with a coverslip, and photograph using a fluorescence microscope.

[0129] The results showed that the comet tail length increased in the tuftolite-treated group, and the tail DNA and tail length were significantly increased, further demonstrating that tuftolite can induce DNA damage. Figure 15 ).

[0130] Example 14 Analysis of BRD4-dependent apoptosis effects

[0131] Four groups were set up: siNC, siBRD4, siNC + coltsfoot ketone, and siBRD4 + coltsfoot ketone. Cells were seeded in 6cm culture dishes and transfected after 16-20 hours. Coltsfoot ketone was added 24 hours later. After 48 hours of treatment, Annexin V / PI flow cytometry was performed.

[0132] The results showed that, under BRD4 knockdown, the increase in apoptosis associated with tuftolone relative to baseline was reduced. Figure 16 ).

[0133] Comprehensive interpretation of experimental results

[0134] The chain of evidence for this invention can be divided into four levels. The first level is candidate findings: SwissTargetPrediction, molecular docking, and molecular dynamics suggest that coltsanthin may bind to BRD4-BD1. The second level is direct target validation: ABPP mass spectrometry, probe-free / UV-free / matrix competition controls, purified BD1 probe labeling, and BLI collectively support direct interaction. The third level is biological effects: coltsanthin inhibits lung adenocarcinoma cell viability, migration, cloning, and DNA synthesis, and promotes apoptosis and DNA damage. The fourth level is target dependence: ShBRD4 attenuates coltsanthin sensitivity in A549 cells, and siBRD4 weakens the additional apoptotic effect of coltsanthin.

[0135] In summary, coltsone can interact with the BRD4-BD1 domain and induce DNA damage, inhibit DNA synthesis, and promote cell death in lung adenocarcinoma cells in a manner that is at least partially dependent on BRD4.

[0136] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of coltsone in the preparation of BRD4 functional inhibitors.

2. The application according to claim 1, characterized in that, The coltsone acts on the BD1 domain of BRD4.

3. The application according to claim 1, characterized in that, The effects of the BRD4 function inhibitor include at least one of the following: It can reduce BRD4-related transcriptional regulatory activity, interfere with BRD4-mediated protein complex or chromatin function, induce BRD4-dependent DNA damage, inhibit DNA synthesis, arrest the cell cycle, or promote tumor cell apoptosis.

4. The application according to claim 3, characterized in that, The induction of BRD4-dependent DNA damage includes at least one of the following: Increased γ-H2AX protein, increased γ-H2AX nuclear focal point, increased comet tail length, increased tail DNA percentage, increased tail moment, or changes in DNA damage response-related proteins.

5. The application according to claim 1, characterized in that, The working concentration of the coltsfoot ketone is 10~80 μM, and the treatment time of the BRD4 function inhibitor is 36~60 h.

6. A BRD4 function inhibitor, characterized in that, It includes coltsone and pharmaceutically acceptable excipients, wherein the mass fraction of coltsone is 0.0001% to 10%; the excipients include at least one of carrier, excipient, stabilizer, solubilizer, emulsifier, thickener, pH adjuster, antioxidant, preservative, buffer, osmotic pressure regulator, sustained-release material or targeted delivery material.

7. The use of coltsfoot ketone in the preparation of drugs for the prevention and / or treatment of diseases related to BRD4 overexpression.

8. The application according to claim 7, characterized in that, The prevention and / or treatment include at least one of the following: increasing γ-H2AX, increasing comet tail moment, reducing cell viability, inhibiting cell proliferation, inhibiting cell migration, inhibiting DNA synthesis, promoting apoptosis, or causing DNA damage.

9. The application according to claim 7, characterized in that, The diseases associated with BRD4 overexpression include lung adenocarcinoma.

10. A drug for the prevention and / or treatment of diseases related to BRD4 overexpression, characterized in that, It includes coltsanthin and pharmaceutically acceptable excipients; wherein the mass fraction of coltsanthin is 0.0001% to 10%; The excipients include at least one of the following: carrier, excipient, stabilizer, cosolvent, emulsifier, thickener, pH adjuster, antioxidant, preservative, buffer, osmotic pressure regulator, sustained-release material, or targeted delivery material; The dosage form of the drug includes at least one of the following: injection, lyophilized powder for injection, liposome, nanoformulation, microsphere formulation, or oral formulation.

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