A dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor and its application in new drug development

Abstract

The present invention relates to the study of biological mechanisms and belongs to the field of biomedicine. The present invention synthesizes a dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor. The probe molecule can simultaneously and accurately target mitochondria and lipid droplet components with a single molecule. It can be used to fluorescently track the physiological activities of lipid droplets and mitochondria during ferroptosis, achieving the increase in lipid droplet polarity, mitochondrial fibrosis, and ONOO during cell ferroptosis. ‑ This technology allows for the visualization and detection of multiple ferroptosis-related biological information, including lipid content, reduced lipid droplet-mitochondrial contact, and gel-like formation of lipid droplets. This solution addresses the inability of ferroptosis-related drugs to report multiple internal microenvironments of lipid droplets and mitochondria, as well as the connection between organelle microdynamics and ferroptosis, at the organelle level, leading to the inability to visualize and evaluate clinical efficacy. It achieves the potential for evaluating the activity of ferroptosis-inducing drugs and provides an accurate screening tool for the development of drugs that induce ferroptosis mechanisms.

Description

Technical Field

[0001] The present invention relates to drug development and mechanism research for disease treatment, and in particular to a coumarin-conjugated bridge-aromatic receptor-based dual-labeled probe and its application in the field of new drug development. Background Art

[0002] Ferroptosis is an iron-dependent, novel type of programmed cell death, distinct from apoptosis, necrosis, and autophagy. The primary mechanism of ferroptosis is the catalysis of highly expressed unsaturated fatty acids on the cell membrane by ferrous iron or esteroxygenase, leading to lipid peroxidation and cell death. Furthermore, ferroptosis is characterized by a decrease in GPX4, the core enzyme regulating the antioxidant system (glutathione system). The main characteristics of ferroptosis include:

[0003] (1) In terms of cell morphology, ferroptosis can cause the size of mitochondria to decrease, membrane density to increase, and cristae to decrease. The behavioral disorders of organelles (lipid droplets and mitochondria) are closely related to the occurrence of ferroptosis.

[0004] (2) In terms of cellular components, ferroptosis is manifested by increased lipid peroxidation and reactive oxygen species. Some characteristic genes also change.

[0005] Ferroptosis sensitivity is closely linked to many biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, as well as the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. It is also associated with pathological cell death associated with mammalian degenerative diseases (e.g., Alzheimer's disease, Huntington's disease, and Parkinson's syndrome), tumors, stroke, cerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and renal failure. Therefore, ferroptosis has become a new direction for drug development, as it can serve as a new type of cell death to kill mesenchymal cells or as an important synergist for immunotherapy and chemotherapy.

[0006] However, the research on ferroptosis in new drug development still faces many technical bottlenecks. The most critical of these is the lack of a toolkit capable of multiplexed reporting on the internal microenvironment of lipid droplets and mitochondria, as well as the connection between organelle microdynamics and ferroptosis. Lipid droplets are inert energy storage sites composed of a polar monolayer (i.e., phospholipids and cholesterol) and non-polar components (i.e., cholesterol esters and triglycerides), forming a liquid-phase organelle. Changes in lipid droplet composition, resulting in changes in polarity, can serve as indicators of their dysfunction. However, the role of controlling the mobility and phase stability of lipid droplet contents in ferroptosis remains unclear. This is especially true for mitochondria, which are energy-providing organelles. Reactive oxygen species can affect mitochondrial appearance, and the stability and dynamic changes in their content levels determine the homeostasis of the mitochondrial microenvironment. In addition to performing their own organelle functions, lipid droplets and mitochondria also exchange material information rapidly and complexly with each other. Given the complexity of this process, using a single indicator to track the effects of drug intervention on ferroptosis does not help researchers understand the specific mechanism of action, which seriously hinders the development of new drugs for potential drugs. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor to solve the problem that ferroptosis-related mechanism drugs cannot multiplex report the internal microenvironment of lipid droplets and mitochondria and the connection between organelle microdynamics and ferroptosis at the organelle level, and cannot be visualized to detect and evaluate clinical efficacy.

[0008] Technical Solution

[0009] A dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor, the structural formula of which includes a coumarin nucleus and an electron receptor, wherein the coumarin nucleus and the electron receptor are connected by a conjugated bridge structure;

[0010] The electron acceptor structure is selected from phenyl derivatives, pyridyl derivatives, and indolyl derivatives;

[0011] The conjugated bridge structure is selected from:

[0012]

[0013] Furthermore, the conjugated bridge structure is The electron acceptor structure is selected from:

[0014]

[0015] Furthermore, the conjugated bridge structure is The electron acceptor structure is selected from:

[0016]

[0017] Furthermore, the conjugated bridge structure is The electron acceptor structure is selected from:

[0018]

[0019] Furthermore, the coumarin core structure is:

[0020]

[0021] Furthermore, the molecular structure of the dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor is:

[0022]

[0023] A method for synthesizing a dual-labeled probe molecule based on coumarin-conjugated bridge-aromatic receptor, comprising the following steps:

[0024]

[0025] Furthermore, the synthesis method includes refluxing 7-(diethylamino)coumarin-3-ethylcarboxylate and 4-hydrazinobenzoic acid in anhydrous ethanol, and obtaining CPC molecules after purification; the molar ratio of the two reaction substrates is 1:0.9~1.1.

[0026] A method for screening drugs that induce ferroptosis, characterized in that the above-mentioned dual-labeled probe molecules that simultaneously target lipid droplets and mitochondria are added to the drug screening system, and the physiological activity indicators of lipid droplets and mitochondria in the process of ferroptosis are simultaneously tracked by fluorescence to obtain the efficacy of the drug in inducing cell ferroptosis.

[0027] Furthermore, the physiological activity indicators include: increased polarity of lipid droplets, mitochondrial fibrosis, ONOO - As the content increases, the lipid droplet-mitochondrial contact decreases and the lipid droplets form a gel-like phase.

[0028] Furthermore, in the drug screening system, the concentration of the dual-labeled probe molecule is 0.1-20.0 μM; the excitation light is selected from 403-407 nm, and the fluorescence emission spectrum is 500-550 nm.

[0029] Beneficial effects

[0030] The present invention synthesized a dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor. The probe molecule can simultaneously and precisely target mitochondria and lipid droplet components with the help of a single molecule, and is used to fluorescently track the physiological activities of lipid droplets and mitochondria during ferroptosis, thereby achieving the increase in lipid droplet polarity, mitochondrial fibrosis, and ONOO during cell ferroptosis. -This technology allows for the visual observation and detection of multiple ferroptosis-related biological signals, including lipid content, reduced lipid droplet-mitochondrial contact, and gel-like formation of lipid droplets. This solution addresses the inability of ferroptosis-related drugs to report multiple internal microenvironments of lipid droplets and mitochondria, as well as the connection between organelle microdynamics and ferroptosis, at the organelle level, leading to the inability to visualize and evaluate clinical efficacy. This technology enables the evaluation of ferroptosis-inducing drug activity in new drug development, providing an accurate screening tool for the development of drugs that induce ferroptosis mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an example of a molecular engineering optical dual-labeling toolbox. (A) The main components of the dual-labeling toolbox's fluorescence response mechanism; (B) The workflow of the dual-labeling toolbox's general synthesis process; (C) A representative tool molecule, CPC, constructed using the dual-labeling toolbox; (D) The interchangeable components of the dual-labeling toolbox.

[0032] Figure 2 This is a characterization of the CPC of the present invention, where the optical response polarity is "on" and responds to ONOO - “Off”. (A) DFT calculations verifying the fluorescence response mechanism of CPC; (B) Mixtures of 1,4-dioxane and water at different ratios (λ ex =458nm) CPC fluorescence emission spectra; (C) different ONOO - CPC fluorescence emission spectra at different concentrations (λ ex =458nm); (D) CPC response polarity "on" and response ONOO - Dual-mode schematic for "off";

[0033] Figure 3 The effects of CPC on lipid droplet polarity and mitochondrial ONOO in living cells are shown in the embodiment of the present invention. - Dual-mode response imaging. (A) SIM image of cells stained with 10 μM CPC for 40 min (scale bar = 5 μm); (B) Merged SIM image of cells stained with CPC and Lipi Blue (scale bar = 5 μm), and magnified image of the area in B (scale bar = 1 μm); (C) Quantitative analysis of the colocalization of CPC and Lipi Blue, data are mean ± SEM (n = 10); (D) Schematic diagram of CPC in lipid droplets; (E) SIM image of CPC in HepG2 cells induced by apocynin (scale bar = 5 μm), and magnified image of the area in E (scale bar = 1 μm); (F) Merged SIM image of CPC and PKMTDR staining after apocynin induction (scale bar = 5 μm), and magnified image of the area in F (scale bar = 1 μm); (G) Quantitative analysis of the colocalization of CPC and PKMTDR, data are mean ± SEM (n = 7); (H) ONOO- SIM images of CPC in HepG2 cells induced by apocynin treated with ONOO (scale bar = 5 μm); (I) untreated and ONOO - Normalized mean fluorescence intensity of CPC in HepG2 cells induced by apocynin treated with ONOO, data are mean ± SEM (n = 10 areas from three cells; ***P < 0.001); (J) untreated and ONOO - Schematic diagram of CPC induced by apocynin in mitochondria; (K) Dual-mode CPC effects on lipid droplet polarity and mitochondrial ONOO in living cells - Schematic diagram of the generated response. (CPC:λ ex =403-407nm, λ em =505–550nm; Lipi Blue: λ ex =403-407nm, λ em =417–476 nm; PKM TDR: λ ex =640nm,λ em =655–705nm).

[0034] Figure 4 This is the mitochondrial ONOO during ferroptosis in the embodiment of the present invention. - Increased lipid droplet polarity. (A) Merged SIM images of cells stained with CPC and PKMTDR in untreated and erastin-treated cells (scale bar = 5 μm). (B) Enlarged images of the indicated areas in A (scale bar = 1 μm). (C) Quantitative analysis of mitochondria in untreated and erastin-treated cells. Data are mean ± SEM (n = 3 cells, *P < 0.05, ****P < 0.0001). (D) Quantitative analysis of colocalization of lipid droplets and mitochondria in untreated and treated cells. Data are mean ± SEM (n = 5 cells, ***P < 0.001). (E) Merged SIM images of cells induced with apocynin and stained with CPC and PKMTDR (scale bar = 5 μm) in untreated and erastin-treated cells; enlarged images of the area in E (scale bar = 1 μm). (F) Fluorescence intensity distribution along white line 1 in E. (G) Fluorescence intensity distribution along white line 2 in E. (H) Size distribution of CPC puncta in untreated and treated cells; data are mean ± SEM (n = 450 puncta from 6 cells). (I) Normalized mean fluorescence intensity of untreated and erastin-treated lipid droplets; data are mean ± SEM (n = 30 fields from 3 cells; ****P < 0.0001). (J) Schematic representation of CPC in untreated and erastin-treated lipid droplets and mitochondria (CPC: λ ex =403-407nm, λ em=505–550nm; PKMTDR: λ ex =640nm,λ em =655–705 nm);

[0035] Figure 5 The formation of a gel-like phase in lipid droplets during ferroptosis in an embodiment of the present invention. (A) SIM image of CPC-stained HepG2 cells (scale bar = 5 μm). (B) SIM image of CPC-stained HepG2 cells after Erastin treatment (scale bar = 5 μm). (C) Enlarged image of the area in A and B (scale bar = 1 μm). (D) Fluorescence intensity distribution along the white arrows in A and B. (E) Schematic diagram of CPC in lipid droplets under untreated and treated conditions. (F) FRAP detection of CPC-stained lipid droplets under untreated and Erastin treatment. (G) Normalized fluorescence recovery curves during the fluorescence recovery process of lipid droplets under untreated and Erastin treatment; data are mean ± SEM (n = 3 measurements). (H) Schematic diagram of FRAP of CPC-stained lipid droplets under untreated and Erastin treatment (CPC: λ ex =403-407nm, λ em =505–550nm);

[0036] Figure 6 Schematic diagram of the CPC molecular action mechanism of the present invention. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0038] General Materials: Ethyl 7-(diethylamino)coumarin-3-carboxylate, 4-hydrazinobenzoic acid, 4-hydrazinopyridine, phenylhydrazine, 4-aminophenylboronic acid, 1,4-butanesultone, and 1,2-dichlorobenzene were all analytical or chemical grade and were not further purified. These synthetic reagents were purchased from MacLean or Aladdin. Fetal bovine serum was purchased from VivaCell (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM #11965118), penicillin-streptomycin (#15140163, 10,000 units / ml), trypsin-EDTA (#25200-072), phenol-free medium (#1894117, Gibco, Grand Island, NY, USA), and other reagents used for cell culture were purchased from Gibco BRL (Grand Island, NY, USA). Erastin, PK Mito-Tracker Deep Red (PKMTDR), and Lipi-Blue were purchased from Invitrogen (Eugene, Oregon, USA). HepG2 cells were provided by Wang Fengshan's laboratory (Shandong University).

[0039] Example 1 Synthesis of a Coumarin-Conjugated Bridge-Aromatic Receptor Dual-Labeled Probe Molecule (CPC)

[0040] A mixture of ethyl 7-(diethylamino)coumarin-3-carboxylate (0.23 g, 0.94 mmol) and 4-hydrazinobenzoic acid (0.143 g, 0.94 mmol) was placed in a 50 mL round-bottom flask. 10 mL of anhydrous ethanol was added and the mixture was refluxed for 3 hours. A brown precipitate formed in the reaction solution. After filtration, the brown precipitate was rinsed with ethyl acetate and dried to yield 0.25 g (0.65 mmol, 69%) of a brown solid.

[0041] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.26 (s, 1H), 10.92 (s, 1H), 8.31 (s, 1H), 8.00 (s, 1H), 7.82 (d, J = 8.8Hz, 2H), 7.59 (d, J = 8.9Hz ,1H),7.11(d,J=8.6Hz,2H),6.74(dd,J=8.9,2.3Hz,1H),6.56(d,J=2.2Hz,1H),3.45(q,J=7.0Hz,4H),1.14(t,J=7.0Hz,6H). 13C NMR(151MHz,DMSO-d6)δ(ppm)(Figure S2):167.71,161.28,156.32,151.12,149.05,136.54,134.02,131.61,130.5 7,120.90,114.48,111.68,110.07,108.89,97.01,44.64,12.85.HRMS(Figure S3):For[M+H] + m / z 380.1610.Found:[M+H] + m / z 379.4800.

[0042] Example 2 Cytotoxicity Assay

[0043] The cytotoxicity was measured by MTT assay. HepG2 cells were plated at 5 × 10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate containing 10% serum DMEM and placed in an incubator (5% CO2, 37°C) for 24 hours. The culture medium was then replaced with 100 μL of fresh DMEM containing different concentrations of CPC (0.1 μM, 1.0 μM, 5.0 μM, 10.0 μM, 20.0 μM and 30.0 μM). After incubation for 12 hours, 20 μL of MTT solution was added to each well and incubated for 4 hours under the same conditions. The culture medium was then removed and added to 150 μL of DMSO. After shaking for 10 minutes, the absorbance of each well at 570 nm was measured using an enzyme-linked immunosorbent assay. Cell viability (%) was calculated by dividing the average absorbance of the experimental group by the average absorbance of the control group.

[0044] Example 3 Cell culture and imaging under OMX 3D-SIM or confocal laser scanning microscope

[0045] HepG2 cells were cultured at 1×10 5The cells were seeded at a density of 100 μM on 35 mm glass-bottomed culture dishes and incubated with 2 ml of DMEM containing 10% serum for 24 h (5% CO2, 37°C). HepG2 cells were incubated with 10.0 μM CPC for 40 min and then washed seven times with fresh DMEM. Finally, HepG2 cells were cultured in 1 ml of phenol-free medium and imaged under an OMX 3D-SIM extended resolution microscope (DeltaVision, Inc) equipped with a 60× / 1.42 numerical aperture oil immersion objective and a solid-state laser. CPC was excited at 403-407 nm and emitted at 500-550 nm, and cell images were acquired at 512×512 using a Z stack with a step size of 0.125 μm. In addition, samples were analyzed under a laser scanning microscope Nikon A1R (Nikon, Japan) equipped with a 63× / 1.4 numerical aperture oil immersion objective.

[0046] Example 4 Flow cytometer analysis

[0047] HepG2 cells were cultured at 5×10 3 Cells were seeded at a density of 100 cells / well in 6-well plates in DMEM with 10% FBS and cultured in an incubator (5% CO2, 37°C) for 24 hours. The culture medium was then replaced with 1000 μL of fresh culture medium containing different concentrations of CPC (0 μM, 0.1 μM, 1.0 μM, 10.0 μM, and 20.0 μM). After staining with CPC for 40 minutes, the cells were trypsinized and washed twice with pre-chilled PBS. The cells were resuspended in 500 μL of buffer and then analyzed by flow cytometry.

[0048] Example 5 Colocalization Experiment

[0049] HepG2 cells were cultured at 1×10 5 HepG2 cells were seeded at a density of 100 μM on a 35 mm glass-bottomed culture dish and incubated with 2 ml of DMEM containing 10% serum for 24 h (5% CO2, 37°C). HepG2 cells were incubated with 10 μM CPC for 40 min, 100 nM PKMTDR for 30 min, and 100 nmol / L Lipi-Blue for 30 min. HepG2 cells were then washed 7 times with fresh DMEM and incubated with 10.0 μM CPC for 40 min. Finally, HepG2 cells were cultured in 1 ml of phenol-free medium and imaged under OMX 3D-SIM. PKMTDR was excited at 640 nm and emitted at 655-705 nm, and Lipi-Blue was excited at 403-407 nm and emitted at 417-476 nm. Images were processed and analyzed using ImageJ.

[0050] Example 6 Data Analysis

[0051] Statistical analysis was performed using Origin 2018, GraphPad Prism 8, and ImageJ. A normality test was used to check for normal distribution. Data are expressed as mean ± standard error of the mean (SEM). The SEM was used to compare experimental results with the control. In the case of non-normal distribution, statistical comparisons of results were tested using the Mann-Whitney test, with the significance level set at ns (no significant difference), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance and sample sizes in all figures and tables are indicated in the corresponding figure legends.

[0052] Example 7 Molecular Engineering Organelle Component Specific Labeling Toolbox

[0053] In order to achieve simultaneous localization in lipid droplets and mitochondria, this technical solution summarizes the structural characteristics of labeled lipid droplets and mitochondria. The lipid droplet labeling structure reflects the following characteristics: the fluorescence of the donor (D)-π-acceptor (A) structure is significantly sensitive to changes in solvent polarity, making it easy to target the internal microenvironment of lipid droplets with low polarity; because coumarin has excellent electron donor properties and its structure is similar to the commercial lipid droplet dye Nile Red, coumarin units are often used as donor components in the design of lipid droplet labeling probes. In addition, the mitochondrial labeling structure takes into account the reactive oxygen species produced by mitochondrial metabolism, especially ONOO - , which can easily destroy the C=N double bond group, therefore, the double bond is often used in the design of mitochondrial labeling probes to respond to ONOO in mitochondria. - .

[0054] With this in mind, this technical solution designed and constructed a dual-labeling toolbox to label lipid droplets and mitochondria. They are composed of a coumarin structure as an electron donor, a double bond structure as a π bridge, and a laboratory-synthesized intermediate as an electron acceptor, forming a typical D–π–A configuration and a fluorescent probe with intramolecular charge transfer (ICT) characteristics. In addition, Group 1 and Group 2 of the structure are replaceable elements that derive different types of π bridges and electron acceptors, respectively ( Figure 1 A). This molecular engineering dual-labeling toolbox uses a one-step condensation reaction of commercially available aldehyde-based coumarins and synthetic intermediates as a general synthetic workflow to derive a series of alternative and rapidly assembled candidate probes ( Figure 1 B) This technical solution assembles a molecular probe from a toolbox based on the elements required for tracking mitochondria and lipid droplets, for example, a coumarin-conjugated bridge-aromatic receptor probe (CPC) constructed by condensation of aldehyde coumarin and carboxyphenylhydrazine under ethanol reflux conditions ( Figure 1C). In addition to CPC, both Group 1 and Group 2 can be replaced with elements in the engineering toolbox to form a systematic combination probe that can be used for engineering screening ( Figure 1 D).

[0055] Example 8 Dual-mode CPC response to in vitro polarity and ONOO -

[0056] Density functional theory (DFT) calculations were first performed to verify the presence of typical ICT features in the coumarin-based probe. The results showed that most of the HOMO electron distribution was located on the carboxylphenylhydrazine unit, while the LUMO was mainly on the aldehyde coumarin ( Figure 2 A), which is consistent with the original intention of designing ICT type probes. Then, CPC was determined to have a Stokes shift of 124nm, which is conducive to obtaining a low signal-to-noise ratio; in fluorescence screening, CPC showed a sensitive fluorescence response to polarity. As the proportion of 1,4-dioxane increased, the fluorescence intensity of CPC gradually increased ( Figure 2 B). This may be because the polarity decreases with the increase of 1,4-dioxane ratio, and the electron-withdrawing ability of polar groups increases, thereby enhancing the ICT effect; the octanol / water partition coefficient (P o / w ) determined the lipophilicity of CPC to be 0.482. Strong fluorescence emission in low-polarity solvents and appropriate lipophilicity and hydrophilicity enable CPC to label lipid droplets. Together, these results demonstrate that CPC possesses a fluorescence "on" function in response to polar environments.

[0057] The π bridge connecting coumarin and the carboxyl group of the benzene ring using a C=N bond is easily affected by ONOO - The maximum absorption wavelength of CPC is about 458nm. When ONOO - When the intensity decreases; in this technical solution, it is observed that when ONOO - When the concentration increases, the fluorescence intensity at 582 nm is quenched ( Figure 2 C). These data were further processed and a lower limit of detection (LOD) of 123 nM was calculated using the formula LOD = 3δ / S, where δ is the standard deviation of 8 blank CPC signals and S is the slope of the calibration curve. 0.1 mM CPC and 0.1 mM ONOO were analyzed using high-resolution mass spectrometry. - The solution after the reaction was confirmed to be CPC-ONOO - Sensing mechanism, 396.9921 mass spectral peak and calculated 397.16338CPC-ONOO - In order to eliminate the influence of potential interference factors, the effect of CPC on Br - 、F - 、H2O2、H2S、K+ In addition, CPC exhibited high fluorescence levels (i.e., >2000 a.u.) in the pH 6 to pH 12 range, which was negative for ONOO. - It exhibits a rapid fluorescence response and only takes about 10 minutes to quench the fluorescence. These experiments show that CPC is a probe with stable fluorescence properties and can respond to ONOO - Overall, when dual-mode is achieved, CPC exhibits excellent in vitro fluorescence properties, namely, responding to polarity "on" and responding to ONOO - Level "off" ( Figure 2 D).

[0058] Example 9 CPC Dual-Mode Imaging Response to Lipid Droplet Polarity and Mitochondrial ONOO in Living Cells - generate

[0059] It was found that CPC has a great effect on ONOO - With sensitive fluorescence response ( Figure 2 C), which is an important aspect of imaging mitochondria. - The fluorescence response was quenched, so the reactive oxygen species inhibitor apocynin was introduced to inhibit ONOO in living cells. - As expected, the SIM images showed the morphology of fluorescent green particles and filamentous mitochondria ( Figure 3 E). In addition, after apocynin induction in HepG2 cells, CPC showed significant overlap with a commercial mitochondrial probe (i.e., PKMTR) ( Figure 3 F), and its PCC showed a suitable value of 0.75 ( Figure 3 G), which confirmed that CPC is localized in mitochondria. - The "off" functional reaction is achieved by adding exogenous ONOO - A control assay was performed ( Figure 3 H); The results showed that the green fluorescence intensity of CPC-labeled mitochondria was significantly reduced ( Figure 3 I), which is due to ONOO in mitochondria - The increase in concentration offsets the effect of the ROS inhibitor, resulting in quenching of green fluorescence ( Figure 3CPC exhibited sufficiently bright fluorescence and efficient cellular uptake at concentrations ranging from 0.1 to 20.0 μM. This protocol examined CPC uptake patterns in HepG2 cells at various temperatures and levels of metabolism and endocytosis, suggesting that CPC enters cells via an energy-dependent mechanism. Finally, this protocol tested CPC's cytotoxicity and observed no effect on cell viability at concentrations up to 30.0 μM.

[0060] In summary, it was concluded in this technical solution that CPC labels lipid droplets and mitochondria in living cells with low toxicity and good cell permeability. In addition, CPC has an effect on the polarity (i.e., opening) of lipid droplets and mitochondrial ONOO in living cells. - Therefore, the use of advanced CPCs can help to gain a more complete understanding of the polarity of organelles and ONOO production during specific diseases. - potential connections between them.

[0061] Example 10 CPC reveals mitochondrial ONOO during ferroptosis - Increased internal polarity of lipid droplets

[0062] Studies have shown that the ferroptosis process involves changes in multiple organelles, and these changes and the regulation of the characteristics of organelle components are becoming an emerging direction for drug design. In this technical solution, the excellent performance of the probe can simultaneously respond to ONOO in mitochondria. - and lipid droplet polarity, inspiring exploration of ONOO in mitochondria during ferroptosis - and changes in lipid droplet polarity.

[0063] In this technical proposal, we initially verified that mitochondrial ONOO during ferroptosis - HepG2 cells were treated with 20.0 μg / mL Erastin for 12 h to induce ferroptosis, and then the cells were stained with CPC and PKMTDR ( Figure 4 A). Compared with the normal state, the morphology and fluorescence intensity of lipid droplets and mitochondria showed significant differences during ferroptosis ( Figure 4 B); In this technical solution, the proportion of elongated mitochondria increased, showing a fibrotic morphology ( Figure 4 C), which is consistent with previous literature reports. In addition, the PCC of lipid droplets and mitochondria decreased from 0.53μM to 0.26μM, which confirmed that Erastin induction caused the disaggregation of lipid droplets and mitochondria ( Figure 4 D).

[0064] To investigate the changes in mitochondrial internal dynamics during lipid droplet-mitochondrial depolymerization, we observed in this protocol that no CPC fluorescence signal was collected on mitochondria during ferroptosis, but was visible in lipid droplets, suggesting that ferroptosis increased ONOO - To further support this assessment, a control experiment was performed in this technical protocol to inhibit the production of reactive oxygen species in HepG2 cells using apocynin and then co-staining CPC and PKMTDR after treatment with or without erastin. Figure 4 E); The results showed that cells without Erastin treatment could collect green fluorescence of CPC-labeled mitochondria ( Figure 4 F), but after treatment of cells with Erastin, the fluorescence was quenched ( Figure 4 G), which indicates that additional ONOO is generated by Erastin - Ferroptosis induction “turns off” CPC fluorescence on mitochondria, which further confirms that mitochondrial morphogenesis forms a contractile-like structure, accompanied by mitochondrial and lipid droplet disaggregation and ONOO - The upregulation of ONOO - levels may be a new avenue for the development of ferroptosis-related drugs.

[0065] In addition to mitochondrial ONOO - In addition to the changes in the lipid droplet size, the size of the lipid droplets was observed to decrease in this technical solution, and the average diameter after Erastin induction was reduced from 0.52 μm to 0.32 μm ( Figure 4 H); It was also noted in this technical protocol that after Erastin treatment, the green fluorescence intensity of CPC-labeled lipid droplets was significantly reduced ( Figure 4 I), which indicates that lipid droplet-mitochondrial disaggregation occurs during ferroptosis, accompanied by a decrease in lipid droplet size and an increase in lipid droplet polarity. In summary, mitochondrial morphological changes and lipid droplet-mitochondrial disaggregation occur during ferroptosis, accompanied by mitochondrial ONOO - Increased levels and increased lipid droplet polarity ( Figure 4 J).

[0066] Example 11: Lipid droplet gel phase during ferroptosis

[0067] In this protocol, CPC was used to demonstrate changes in lipid droplet-mitochondrial interactions during ferroptosis. - This is attributed to intracellular lipid peroxidation during ferroptosis, in which the damaged mitochondrial inner membrane forms a fibrillar morphology and produces a large amount of ONOO -, which leads to quenching of green fluorescence on CPC-labeled mitochondria. However, it is worth noting that the mechanism by which the increase in lipid droplet polarity leads to a decrease in lipid droplet size remains unclear. Lipid droplets are inert energy storage sites composed of a polar monolayer (i.e., phospholipids and cholesterol) and non-polar components (i.e., cholesterol esters and triglycerides); because these multiple components form the polarized lipid droplet environment and determine lipid droplet size, it is reasonable to speculate that the polarized material components form a homogeneous system during ferroptosis.

[0068] When the phase separation elements are inactivated, a gel-like phase emerges in the liquid environment, which is a recently reported evolutionarily conserved mechanism that aggregates the non-functional gel-like phase to facilitate better clearance. We next examined CPC-stained lipid droplets in the absence and treatment with Erastin ( Figure 5 A and 5B). In the untreated group, the uneven fluorescence intensity distribution formed a fluorescence collapse, indicating that the content of lipid droplets was uneven ( Figure 5 C and 5D). This fluorescence collapse disappeared after Erastin treatment, indicating that the different polarized substances inside the lipid droplets continued to decrease and gradually formed non-metabolized components with relatively uniform polarity, resulting in the disappearance of fluorescence collapse ( Figure 5 E). To further confirm this statement, a fluorescence recovery after photobleaching (FRAP) experiment was carried out in this technical scheme ( Figure 5 F) to study the fluidity inside lipid droplets. It was found that CPC fluorescence recovery in normal lipid droplets was faster than that during ferroptosis, indicating that during ferroptosis, lipid droplets formed a non-functional gel-like phase ( Figure 5 G). This gel-like phase is a recently discovered physiological process that typically occurs in damaged mtDNA, during which non-functional gel-like aggregates form to facilitate better clearance. In this technical scheme, it was found that during ferroptosis, a gel-like phase forms in the contents of lipid droplets, indicating an initial loss of lipid droplet function ( Figure 5 H), which provides a new perspective for the development of lipid metabolism-related drugs.

[0069] Results and discussion

[0070] The present invention provides a molecular engineering toolbox probe for dual-labeling CPC in lipid droplets and mitochondria, and nanoscale fluorescence tracking of specific components of lipid droplets and mitochondria during ferroptosis. CPC has been shown to be a cytosolic inhibitor of ONOO in mitochondria. - CPC is an excellent tool for dual responses to lipid droplet polarity, revealing the formation of fibrillar mitochondria and a gel-like phase in lipid droplets, which inhibits lipid droplet-mitochondrial interactions during ferroptosis. Thus, CPC addresses the urgent need for multiplexed reporting of the changing microdynamics of lipid droplets and mitochondria and reveals a link between organelle microdynamics and ferroptosis.

[0071] Ferroptosis is a newly discovered type of cell death characterized by lipid peroxidation associated with various organelle components. It plays a role in a variety of diseases such as cancer, neurodegenerative diseases, and inflammation, and has become a new direction for drug development. However, due to the extreme complexity of the ferroptosis process, there are technical bottlenecks, including the lack of tools for multiplex reporting of organelle microdynamics and the lack of tools to explain the connection between organelle microdynamics and ferroptosis, which seriously hinders the exploration of potential drugs. To eliminate these obstacles, in this technical protocol, CPC is combined with super-resolution SIM imaging technology to study the microscopic dynamic changes of lipid droplets and mitochondria in normal states and during ferroptosis; this enables the observation in this technical protocol that increasing the polarity of lipid droplets leads to the appearance of shrunken lipid droplets, forming a non-functional gel-like phase, and upregulating mitochondrial ONOO - The content triggers the formation of fibrillar mitochondria during ferroptosis, which supports the changes in organelle microdynamics. The phase separation behavior of cellular molecules has been revealed as a key mechanism, such as P particles, mTOR, SHP2, synaptosomes, P62, nuclear phosphoprotein nuclear DNA, fused RNA in sarcoma, binding protein (FUS), etc., indicating that phase separation can also be used as one of the directions for drug development. The use of the present invention further reveals the formation of a gel-like phase in lipid droplets and fibrillar mitochondria, inhibiting lipid droplet-mitochondrial interactions during ferroptosis, which confirms the connection between organelle microdynamics during ferroptosis.

[0072] Overall, the findings of this study provide important insights into the dynamic changes in the microenvironment of mitochondria and lipid droplets, and open up new avenues for the potential design of drugs focused on the diagnosis and treatment of specific organelle components during ferroptosis. In summary, CPC will be able to conduct a deeper study of the microdynamics of different organelles during ferroptosis, which can then be expanded to the study of other cellular states, providing underlying technical support for the development of drugs that induce ferroptosis.

Claims

1. A dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor, characterized in that: The structural formula includes a coumarin nucleus and an electron acceptor, wherein the coumarin nucleus and the electron acceptor are connected via a conjugated bridge structure; The electron acceptor structure is selected from phenyl derivatives, pyridyl derivatives, and indolyl derivatives; The conjugated bridge structure is selected from: The conjugated bridge structure is: When the electron acceptor structure is selected from: The conjugated bridge structure is: When the electron acceptor structure is selected from: The conjugated bridge structure is: When the electron acceptor structure is selected from:

2. The dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor according to claim 1, characterized in that: The coumarin core structure is:

3. The dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor according to claim 2, characterized in that: The molecular structure of the dual-labeled probe based on coumarin-conjugated bridge-aromatic receptor is:

4. A method for synthesizing a dual-labeled probe molecule based on coumarin-conjugated bridge-aromatic receptor, characterized in that: The steps include, 5. The method for synthesizing a dual-labeled probe molecule based on coumarin-conjugated bridge-aromatic receptor according to claim 4, characterized in that: The steps include: subjecting 7-(diethylamino)coumarin-3-ethylcarboxylate and 4-hydrazinobenzoic acid to a reflux reaction in anhydrous ethanol, and obtaining CPC molecules after purification; the molar ratio of the two reaction substrates is 1:0.9-1.

1.

6. A method for screening drugs that induce ferroptosis, characterized in that: The dual-labeled probe molecule according to any one of claims 1 to 3 that simultaneously targets lipid droplets and mitochondria is added to the drug screening system, and the physiological activity indicators of lipid droplets and mitochondria in the process of ferroptosis are simultaneously tracked by fluorescence to obtain the efficacy of the drug in inducing cell ferroptosis.

7. The method for screening a drug that induces ferroptosis according to claim 6, wherein: The physiological activity indicators include: increased polarity of lipid droplets, mitochondrial fibrosis, ONOO - As the content increases, the lipid droplet-mitochondrial contact decreases and the lipid droplets form a gel-like phase.

8. The method for screening a drug that induces ferroptosis according to claim 6, wherein: The concentration of the dual-labeled probe molecule in the drug screening system is 0.1-20.0 μM.

9. The method for screening a drug that induces ferroptosis according to claim 6, wherein: The excitation light is selected from 403-407 nm, and the fluorescence emission spectrum is 500-550 nm.

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