A targeted ferroptosis-inducing conjugate and a preparation method and application thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a targeted ferroptosis-inducing conjugate as well as a preparation method and application thereof. The structural formula of the conjugate is as follows: the conjugate contains a prostate cancer targeting peptide segment WHDGFK, and is connected with an iron death-inducing antibacterial peptide KRIVKWIIKLLR through a disulfide bond, so that the conjugate has tumor targeting and microenvironment response release functions, not only endows the antibacterial peptide KRIVKWIIKLLR with tumor selective delivery capacity, but also realizes specific release of the active peptide in target cells by reducing the disulfide bond by using high-concentration glutathione (GSH) in tumor cells, thereby synergistically enhancing the antitumor effect and reducing the systemic toxicity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a targeted ferroptosis-inducing conjugate, its preparation method, and its application. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors of the male reproductive system, and its incidence is increasing globally. Although early-stage prostate cancer can achieve good prognoses through surgery, radiotherapy, or endocrine therapy, the treatment of advanced and castration-resistant prostate cancer (CRPC) still faces significant challenges. Currently used chemotherapy drugs such as docetaxel and cabazitaxel have some efficacy, but their non-specific distribution leading to systemic toxicity, drug resistance, and a narrow therapeutic window severely limits their long-term application. Therefore, developing novel targeted therapies with high selectivity and low toxicity has become a hot topic in prostate cancer research.

[0003] In recent years, antimicrobial peptides (AMPs) have attracted widespread attention due to their broad-spectrum antimicrobial activity and unique membrane disruption mechanisms. Increasing research indicates that some antimicrobial peptides also exhibit significant killing effects on tumor cells, with mechanisms including not only cell membrane perforation but also induction of apoptosis, inhibition of angiogenesis, and regulation of immune responses. KRIVKWIIKLLR is a peptide with potent antimicrobial activity (Antibacterial and Anti-Inflammatory Properties of a Novel Antimicrobial Peptide Derived from LL-37, Antibiotics (Basel, Switzerland) 2022 Vol.11 No.6 P754 2079-6382). Previous studies have found that it can induce tumor cell apoptosis by promoting ferroptosis, demonstrating potential antitumor applications. However, this peptide lacks tumor specificity and is easily non-specifically taken up by normal tissues after systemic administration, leading to decreased efficacy and potential toxic side effects, severely limiting its in vivo application.

[0004] Ferroprelation is a newly discovered programmed cell death mechanism in recent years, characterized by the accumulation of iron-dependent lipid peroxides. It differs significantly from classic cell death mechanisms such as apoptosis, necrosis, and autophagy in morphology, biochemistry, and genetic mechanisms. Studies have shown that inducing ferroptosis in tumor cells is a novel strategy to overcome resistance to traditional chemotherapy and enhance the efficacy of immunotherapy. However, achieving tumor-specific delivery of ferroptosis inducers remains a challenge and a hot research topic.

[0005] Tumor-targeting peptides have become an important component of tumor-targeted delivery systems due to their advantages such as small molecular weight, low immunogenicity, strong tissue penetration, and ease of modification. WHDGFK is a prostate cancer-specific binding peptide obtained through phage display screening. It can specifically bind to prostate-specific membrane antigen (PSMA), which is highly expressed on the surface of prostate cancer cells, mediating the selective enrichment of its conjugates in tumor tissue. Conjugating targeting peptides with antitumor active molecules through chemical bonds to construct tumor microenvironment-responsive drug delivery systems is one of the ideal strategies for achieving precision medicine; however, research on this type of conjugation is currently scarce. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that existing active peptides are difficult to achieve specific release in target cells, resulting in poor anti-tumor effects. In order to overcome the above-mentioned defects of the prior art, the present invention provides a targeted ferroptosis-inducing conjugate, its preparation method and application.

[0007] A first aspect of the present invention provides a targeted ferroptosis-inducing conjugate, characterized in that it has the following structural formula: .

[0008] Compared with existing technologies, this conjugate contains the prostate cancer-targeting peptide WHDGFK, which is linked to the ferroptosis-inducible antimicrobial peptide KRIVKWIIKLLR via a disulfide bond. This gives the conjugate tumor-targeting and microenvironment-responsive release capabilities, not only endowing the antimicrobial peptide KRIVKWIIKLLR with tumor-selective delivery capabilities, but also utilizing the high concentration of glutathione (GSH) in tumor cells to reduce disulfide bonds, thereby achieving the specific release of the active peptide into target cells, thus synergistically enhancing its antitumor effect and reducing systemic toxicity.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned targeted ferroptosis-inducing conjugate, comprising the following steps: S1. Dissolve the peptide WHDGFK in anhydrous N,N-dimethylformamide. Under ice bath cooling, add 3,3'-dithiodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine in sequence. Stir at room temperature. After the reaction is complete, add dilute hydrochloric acid and continue stirring until the pH is neutral. Extract the organic phase from the reaction product, and obtain the intermediate WHD-SS by drying, vacuum concentration and purification. S2. Dissolve the intermediate WHD-SS in anhydrous N,N-dimethylformamide, add peptide KRIVWIIKLLR, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine, stir at room temperature until the reaction is complete, then add dilute hydrochloric acid and continue stirring until the pH is neutral, extract the organic phase in the reaction product, and obtain the targeted ferroptosis-inducing conjugate by drying, vacuum concentration and purification. The structural formula of the peptide WHDGFK is: .

[0010] In one possible implementation, the molar ratio of peptide WHDGFK to 3,3'-dithiodipropionic acid in step S1 is between 1:1 and 1:1.5.

[0011] In one possible implementation, the stirring time at room temperature in step S1 is 8-16 h, and the stirring time is continued for 0.5-2 h after adding dilute hydrochloric acid.

[0012] In one possible implementation, the molar ratio of intermediate WHD-SS to peptide KRIVWIIKLLR in step S2 is between 1:1 and 1:1.5.

[0013] In one possible implementation, the stirring time at room temperature in step S2 is 12-24 h, and the stirring time is continued for 0.5-2 h after adding dilute hydrochloric acid.

[0014] In one possible implementation, the extractant used in both steps S1 and S2 is dichloromethane.

[0015] In one possible implementation, anhydrous sodium sulfate is used as a desiccant for drying in both steps S1 and S2.

[0016] In one possible implementation, the concentration of dilute hydrochloric acid used in steps S1 and S2 is 1 mol / L.

[0017] A third aspect of the present invention is to provide the use of the above-described targeted ferroptosis-inducing conjugate in the preparation of a drug for treating prostate cancer.

[0018] The beneficial effects of this invention are as follows: 1. WHDGFK is a prostate cancer-specific binding peptide obtained through phage display screening. It can specifically bind to prostate-specific membrane antigen (PSMA) that is highly expressed on the surface of prostate cancer, mediating the selective enrichment of its conjugate in tumor tissue.

[0019] 2. By chemically coupling the targeting peptide with antitumor active molecules, a tumor microenvironment-responsive drug delivery system is constructed, enabling the use of prostate cancer targeting peptides and ferroptosis-inducing antimicrobial peptides coupled via disulfide bonds for targeted and precise treatment of prostate cancer.

[0020] 3. The novel targeted therapy strategy constructed by this conjugate has high selectivity and low toxicity. Attached Figure Description

[0021] Figure 1 The synthetic route for compound WHD-SS-KRI in Example 1 is shown below; Figure 2 The hydrogen NMR spectrum (DMSO, 500 MHz) of the intermediate WHD-SS in Example 1. Figure 3 The 1H NMR spectrum (DMSO, 500 MHz) of compound WHD-SS-KRI in Example 1 is shown. Figure 4 Comparison of uptake of free KRIVKWIIKLLR and WHD-SS-KRI in RM-1 cells; Figure 5 The inhibitory effects of WHD-SS-KRI and free KRIVKWIIKLLR on the proliferation of RM-1 cells; Figure 6 Intracellular lipid peroxidation levels were detected by 581 / 591 C11 fluorescence staining with PBS, free KRIVKWIIKLLR and WHD-SS-KRIBODIPY; Figure 7 Quantitative determination of intracellular GSH content in PBS, free KRIVKWIIKLLR and WHD-SS-KRIBODIPY; Figure 8 FerroOrange fluorescence staining with PBS, free KRIVKWIIKLLR and WHD-SS-KRIBODIPY to detect intracellular Fe 2+ level; Figure 9 Western blotting of PBS, free KRIVKWIIKLLR and WHD-SS-KRIBODIPY was used to detect GPX4 protein expression levels and perform semi-quantitative analysis. Detailed Implementation

[0022] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0023] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0025] This invention discloses a method for preparing a targeted ferroptosis-inducing conjugate, comprising the following steps: S1. Dissolve the peptide WHDGFK in anhydrous N,N-dimethylformamide, and add 3,3'-dithiodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine sequentially under ice bath cooling. Stir at room temperature. After the reaction is complete, add dilute hydrochloric acid and continue stirring until the pH is neutral. Extract the organic phase from the reaction product, and obtain the intermediate WHD-SS by drying, vacuum concentration and purification. S2. Dissolve the intermediate WHD-SS in anhydrous N,N-dimethylformamide, add peptide KRIVWIIKLLR, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine, stir at room temperature until the reaction is complete, then add dilute hydrochloric acid and continue stirring until the pH is neutral, extract the organic phase in the reaction product, and obtain the targeted ferroptosis-inducing conjugate by drying, vacuum concentration and purification. The structural formula of the peptide WHDGFK is: .

[0026] In step S1, the molar ratio of peptide WHDGFK to 3,3'-dithiodipropionic acid is between 1:1 and 1:1.5. In step S1, the stirring time at room temperature is 8–16 h, and the stirring time continues for 0.5–2 h after adding dilute hydrochloric acid.

[0027] In step S2, the molar ratio of intermediate WHD-SS to peptide KRIVWIIKLLR is between 1:1 and 1:1.5. In step S2, the stirring time at room temperature is 12–24 h, and after adding dilute hydrochloric acid, the stirring time is continued for 0.5–2 h.

[0028] Dichloromethane was used as the extractant in both steps S1 and S2. Anhydrous sodium sulfate was used as the drying agent in both steps S1 and S2. The concentration of dilute hydrochloric acid used in both steps S1 and S2 was 1 mol / L.

[0029] The structural formula of the obtained targeted ferroptosis-inducing conjugate is as follows: .

[0030] This targeted ferroptosis-inducing conjugate can be used to prepare drugs for the treatment of prostate cancer.

[0031] Example 1 The synthesis and structural characterization of a targeted ferroptosis-inducible conjugate (denoted as WHD-SS-KRI) specifically include the following steps: (1) Synthesis of intermediate WHD-SS In a dry 100 mL round-bottom flask, the peptide WHDGFK (0.5 mmol, approximately 344 mg) was dissolved in 20 mL of anhydrous N,N-dimethylformamide. Under ice-bath cooling, 3,3'-dithiodipropionic acid (0.5 mmol, 105 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.6 mmol, 115 mg), and then N,N-diisopropylethylamine (DIPEA, 0.6 mmol, 77 mg) were added dropwise. EDCI and DIPEA served as catalysts. After the addition was complete, the reaction mixture was moved to room temperature and stirred for 16 h. The reaction was monitored by TLC. After the reaction was completed, 50 mL of dilute hydrochloric acid (0.1 M) was added and the mixture was stirred for 0.5 h. The mixture was then extracted with dichloromethane (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (mobile phase: acetonitrile / water) to obtain a white solid WHD-SS (260 mg, yield 53%).

[0032] (2) Synthesis of compound WHD-SS-KRI Compound WHD-SS (0.1 mmol, approximately 98 mg) was dissolved in 20 mL of anhydrous N,N-dimethylformamide and added to a 50 mL round-bottom flask. Peptide KRIVWIIKLLR (0.1 mmol, approximately 182 mg), EDCI (0.12 mmol, 23 mg), and DIPEA (0.12 mmol, 15 mg) were added, and the mixture was stirred at room temperature for 24 h. The reaction was monitored by HPLC. After the reaction was complete, 50 mL of dilute hydrochloric acid (0.1 M) was added, and stirring continued for 0.5 h. The mixture was then extracted with dichloromethane (3 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative high-performance liquid chromatography (mobile phase: acetonitrile / water) to obtain a white solid WHD-SS-KRI (69 mg, yield 25%).

[0033] The compound WHD-SS obtained in step (1) was subjected to 1H NMR spectroscopy, and the results are as follows: Figure 2As shown, the vibrational response corresponding to the 1H NMR data in the figure is as follows: 1H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 12.02 (s, 1H), 11.86 (s, 1H), 10.76 (d, J = 8.3 Hz, 1H), 9.22 (dd, J = 7.6, 6.6 Hz, 1H), 8.39 (dd, J = 7.6, 2.0 Hz, 1H), 8.29 (d, J = 11.2 Hz, 1H), 8.22 (d, J = 10.7 Hz, 1H), 8.12 (dd, J = 11.7, 2.2 Hz, 2H), 8.07 – 7.99 (m, 1H), 7.64 (dt, J = 7.5, 1.2 Hz, 1H), 7.55 (t, J = 6.7 Hz, 1H), 7.38 (td, J = 6.8, 1.9 Hz,2H), 7.34 – 7.26 (m, 5H), 7.30 – 7.22 (m, 1H), 7.17 (td, J = 7.4, 1.5 Hz, 1H), 7.12 (td, J = 7.3, 1.6 Hz, 1H), 6.54 (t, J = 7.9 Hz, 1H), 6.19 (t, J =7.9 Hz, 1H), 4.67 (dt, J = 11.0, 7.0 Hz, 1H), 4.50 (ddt, J = 17.7, 11.0, 7.1Hz, 2H), 4.15 – 4.08 (m, 1H), 4.11 – 4.03 (m, 2H), 4.04 (s, 1H), 3.25 (dd, J= 12.3, 7.0 Hz, 1H), 3.20 – 3.14 (m, 1H), 3.17 – 3.09 (m, 5H), 3.13 – 3.07(m, 2H), 3.09 – 3.00 (m, 3H), 2.98 (dd, J = 12.5, 7.1 Hz, 1H), 2.83 – 2.69(m, 2H), 2.63 (d, J = 6.9 Hz, 2H), 2.64 – 2.49 (m, 2H), 1.67 (dq, J = 12.3, 7.0 Hz, 1H), 1.63 – 1.37 (m, 4H), 1.31 (dq, J = 12.2, 7.0 Hz, 1H). This confirms the structural formula of the peptide WHDGFK as follows: .

[0034] The compound WHD-SS-KRI obtained in step (2) was subjected to 1H NMR spectroscopy, and the results are as follows: Figure 3As shown, the vibrational responses corresponding to the 1H NMR data in the figure are as follows: 1H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 12.11 (s, 1H), 12.02 (s, 1H), 11.93 (s, 1H), 10.88 (s, 1H), 10.74 (d, J = 2.7 Hz, 2H), 8.31 (d, J = 1.9 Hz, 1H), 8.17 (s, 1H), 8.15 – 8.09 (m, 13H), 8.07 – 8.00 (m, 3H), 7.97 (s, 1H), 7.64 – 7.53 (m, 6H), 7.37 (d, J = 2.0 Hz, 1H), 7.32 (dt, J = 7.2, 1.8 Hz, 2H), 7.29 – 7.16 (m, 9H), 7.16 – 7.07 (m, 4H), 7.10 – 7.04 (m, 1H), 6.99 (s, 1H), 6.84 (d, J = 10.0 Hz, 3H), 6.53 (s, 1H), 6.18 (s, 2H), 4.56 (dt, J = 9.0, 7.1 Hz, 2H), 4.45 – 4.29 (m, 8H), 4.26 (d, J= 6.6 Hz, 2H), 4.21 – 4.13 (m, 2H), 4.16 – 4.11 (m, 1H), 4.10 – 4.03 (m, 2H),4.05 – 3.95 (m, 3H), 3.91 (t, J = 7.1 Hz, 1H), 3.38 (t, J = 6.9 Hz, 1H), 3.20(t, J = 7.2 Hz, 4H), 3.13 – 2.93 (m, 18H), 2.84 (dd, J = 7.0, 1.6 Hz, 2H),2.64 – 2.50 (m, 10H), 2.03 (dq, J = 13.7, 6.8 Hz, 1H), 1.96 (s, 3H), 1.84(dtd, J = 13.8, 6.9, 0.9 Hz, 3H), 1.75 – 1.19 (m, 47H), 0.90 – 0.74 (m, 35H). Therefore, the structural formula of compound WHD-SS-KRI is: Its synthetic route is as follows Figure 1 As shown.

[0035] Example 2 Tumor targeting evaluation of compound WHD-SS-KRI obtained in Example 1 To further validate the targeting ability of WHD-SS-KRI to prostate cancer cells, mouse prostate cancer cell line RM-1 was cultured in RPMI 1640 medium containing 10% fetal bovine serum and routinely passaged at 37℃ and 5% CO2. Logarithmically growing cells were seeded into 6-well plates. When cell confluence reached 80%, equimolar concentrations (50 μM) of free KRIVKWIIKLLR or the conjugate WHD-SS-KRI were added, and incubation continued at 37℃ for 6 h. After incubation, the culture medium was discarded, and cells were washed three times with pre-cooled PBS (phosphate-buffered saline) to remove unbound peptides. Cell lysis buffer (acetonitrile-water (1:1, containing 0.1% trifluoroacetic acid)) was added to lyse cells and precipitate proteins. After sonication-assisted extraction, the cells were centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. Quantitative analysis was performed using high-performance liquid chromatography (HPLC). The results are as follows: Figure 4 The results showed that the relative intracellular uptake of the WHD-SS-KRI group was 5.83±0.32, significantly higher than that of the free KRIVKWIIKLLR group (1.09±0.08), approximately 5.4 times higher. This indicates that the introduction of the prostate cancer-targeting peptide WHDGFK significantly enhances the accumulation of the antimicrobial peptide KRIVKWIIKLLR in RM-1 cells, confirming the good tumor cell targeting properties of this conjugate.

[0036] Example 3 Evaluation of the antitumor activity of compound WHD-SS-KRI obtained in Example 1 The free antimicrobial peptide KRIVKWIIKLLR was used as a control. RM-1 cells in logarithmic growth phase were used, with 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 mg / L in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. The culture medium was discarded, and fresh culture medium containing different concentrations of WHD-SS-KRI or KRIVKWIIKLLR was added to each well, with 5 replicates per concentration. Incubation continued for 48 h. After incubation, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 h. The absorbance of each well was measured at 450 nm. With the cell viability of the untreated group set at 100%, the cell viability of each treatment group was calculated. Nonlinear regression analysis was performed using GraphPad Prism 8.0 software to calculate the half-maximal inhibitory concentration (IC50). 50 The results showed that the inhibitory effect of compound WHD-SS-KRI on the proliferation of RM-1 cells was significantly dose-dependent, with an IC50 value of [missing information]. 50The value is 53.36 nM; while the IC of free KRIVKWIIKLLR is... 50 The value is 132.4 nM, approximately 2.5 times that of the former. Figure 5 This result indicates that the introduction of the prostate cancer-targeting peptide WHDGFK significantly enhances the in vitro antitumor activity of the antimicrobial peptide KRIVKWIIKLLR against RM-1 cells, consistent with its mechanism of targeting and enhancing cellular uptake.

[0037] Example 4 Mechanism of ferroptosis induced by the compound WHD-SS-KRI obtained in Example 1 in prostate cancer cells The level of lipid reactive oxygen species (lipid peroxidation) in RM-1 cells treated with WHD-SS-KRI was detected using the BODIPY 581 / 591 C11 fluorescent probe. RM-1 cells were seeded in confocal culture dishes and cultured for 24 h. Then, equimolar concentrations (50 nM) of free KRIVKWIIKLLR or WHD-SS-KRI were added and incubated for 12 h, with a PBS control group included. After incubation, 2 μM of BODIPY 581 / 591 C11 probe was added and stained at 37℃ in the dark for 30 min. After washing with PBS, the cells were observed under a confocal microscope (excitation wavelength 488 nm / 581 nm, emission wavelength 510 nm / 610 nm). The results showed that cells in the PBS control group exhibited strong red fluorescence (reduced state) and weak green fluorescence (oxidized state); some green fluorescence was observed in the KRIVKWIIKLLR group, indicating that free peptides can induce a certain degree of lipid peroxidation; while the green fluorescence of cells in the WHD-SS-KRI group was significantly enhanced, the red fluorescence was significantly weakened, and the red-green fluorescence ratio was significantly lower than that of the KRIVKWIIKLLR group. Figure 6 This result indicates that WHD-SS-KRI can more effectively induce the accumulation of lipid peroxides in RM-1 cells, consistent with typical characteristics of ferroptosis.

[0038] The intracellular GSH content in RM-1 cells of each group was detected using a micro-reduced glutathione assay kit. RM-1 cells in logarithmic growth phase were seeded in 6-well plates and incubated for 12 h with equimolar concentrations (50 nM) of free KRIVKWIIKLLR or WHD-SS-KRI, with a PBS-treated group serving as a control. After cell collection and precipitation, absorbance was measured at 405 nm according to the kit instructions. The intracellular GSH content (μmol / g prot) was calculated by normalizing the protein concentration determined by the BCA method. Results showed ( Figure 7The intracellular GSH content in the PBS control group was 47.94 ± 1.15 μmol / g prot; the GSH content in the KRIVKWIIKLLR group decreased to 34.39 ± 1.95 μmol / g prot (p < 0.01 vs PBS group); while the GSH content in the WHD-SS-KRI group was significantly reduced to 13.34 ± 1.10 μmol / g prot, showing a highly significant difference compared to both the PBS and KRIVKWIIKLLR groups. These results indicate that WHD-SS-KRI directly consumes GSH through disulfide bond-mediated GSH-responsive cleavage, and the released bioactive peptides induce ferroptosis, further exacerbating GSH depletion.

[0039] FerroOrange fluorescent probes were used to detect intracellular ferrous ions (Fe2+) in RM-1 cells of each group. 2+ Cells were treated as above, and after incubation, 1 μM FerroOrange probe was added and stained at 37°C in the dark for 30 min. After washing with PBS, the cells were observed under a confocal microscope (excitation wavelength 561 nm, emission wavelength 580-630 nm). The results showed that ( Figure 8 Weak fluorescence was observed in cells of the PBS control group, indicating basal Fe levels. 2+ The fluorescence intensity of the KRIVKWIIKLLR group was slightly enhanced; while the WHD-SS-KRI group showed strong orange fluorescence, with a significantly higher fluorescence intensity than the KRIVKWIIKLLR group. This result indicates that WHD-SS-KRI can more effectively promote intracellular Fe in RM-1 cells. 2+ The accumulation of iron ions provides the necessary source of iron ions for ferroptosis to occur.

[0040] The protein expression level of glutathione peroxidase 4 (GPX4) in RM-1 cells of each group was detected by Western blotting. Cell treatment was the same as above. After incubation, cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by BCA method. An equal amount of protein (30 μg) was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk powder, GPX4 primary antibody (1:1000) and GAPDH primary antibody (1:5000) were added and incubated overnight at 4℃. HRP-labeled secondary antibody was incubated at room temperature for 1 h. Development was performed using ECL chemiluminescence, and grayscale analysis was performed using ImageJ software. The relative expression level of GPX4 protein was calculated using GAPDH as an internal control. The results showed ( Figure 9The relative expression level of GPX4 protein in the PBS control group was 0.606±0.053; in the KRIVKWIIKLLR group, GPX4 expression decreased to 0.445±0.075; while in the WHD-SS-KRI group, GPX4 expression was significantly downregulated to 0.144±0.012, showing highly significant differences compared to both the PBS and KRIVKWIIKLLR groups. GPX4 is a key negative regulator of ferroptosis, and its downregulation further confirms the mechanism by which WHD-SS-KRI exerts its antitumor effect by inducing ferroptosis.

[0041] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A targeted ferroptosis-inducing conjugate, characterized in that, The structure is as follows: 。 2. A method for preparing the targeted ferroptosis-inducing conjugate according to claim 1, characterized in that, Includes the following steps: S1. Dissolve the peptide WHDGFK in anhydrous N,N-dimethylformamide. Under ice bath cooling, add 3,3'-dithiodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine in sequence. Stir at room temperature. After the reaction is complete, add dilute hydrochloric acid and continue stirring until the pH is neutral. Extract the organic phase from the reaction product, and obtain the intermediate WHD-SS by drying, vacuum concentration and purification. S2. Dissolve the intermediate WHD-SS in anhydrous N,N-dimethylformamide, add peptide KRIVWIIKLLR, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine, stir at room temperature until the reaction is complete, then add dilute hydrochloric acid and continue stirring until the pH is neutral, extract the organic phase in the reaction product, and obtain the targeted ferroptosis-inducing conjugate by drying, vacuum concentration and purification. The structural formula of the peptide WHDGFK is: .

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of peptide WHDGFK to 3,3'-dithiodipropionic acid is between 1:1 and 1:1.

5.

4. The preparation method according to claim 2, characterized in that, In step S1, the stirring time at room temperature is 8~16 h, and the stirring time is continued for 0.5~2 h after adding dilute hydrochloric acid.

5. The preparation method according to claim 2, characterized in that, In step S2, the molar ratio of intermediate WHD-SS to peptide KRIVWIIKLLR is between 1:1 and 1:1.

5.

6. The preparation method according to claim 2, characterized in that, In step S2, the stirring time at room temperature is 12~24 h, and the stirring time is continued for 0.5~2 h after adding dilute hydrochloric acid.

7. The preparation method according to claim 2, characterized in that, The extractant used in both steps S1 and S2 is dichloromethane.

8. The preparation method according to claim 2, characterized in that, Anhydrous sodium sulfate was used as a drying agent in both steps S1 and S2.

9. The preparation method according to claim 2, characterized in that, The concentration of dilute hydrochloric acid used in steps S1 and S2 is 1 mol / L.

10. The use of the targeted ferroptosis-inducing conjugate of claim 1 in the preparation of a drug for treating prostate cancer.

Images