GPX4 protein targeted degradation agent as well as preparation method and application thereof

Abstract

The invention relates to a GPX4 protein targeted degradation agent as well as a preparation method and application thereof. The GPX4 protein targeting degradation agent comprises a tumor cell targeting unit, an ROS response unit, an E3 ligase recognition unit, a self-assembly unit and a GPX4 targeting binding unit which are connected in sequence, the tumor cell targeting unit comprises a ligand of an epidermal growth factor receptor. The GPX4 protein targeted degradation agent can efficiently degrade target protein GPX4 in a high-concentration active oxygen environment in tumor cells so as to induce ferroptosis of the tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a GPX4 protein-targeting degrader, a preparation method thereof, and an application thereof. Background Art

[0002] The targeted protein degradation technology can directly target abnormal proteins, which are the core factors in the occurrence and development of diseases, and is crucial for the treatment of various diseases. Most diseases (such as cancer) are related to abnormal protein expression or dysfunction. Traditional drug research and development focuses on small molecule inhibitors, which inhibit the function of target proteins by occupying the active sites of target proteins. However, this method relies on the existence of a bindable active pocket in the target protein and requires maintaining a high drug concentration for continuous inhibition, which easily leads to off-target effects, drug resistance, and toxicity. In addition, many key target proteins (such as GPX4) are difficult to develop effective inhibitors because they lack a "druggable" pocket in the traditional sense. Based on the disadvantages of small molecule inhibitors, the proteolysis-targeting chimera (PROTAC) technology has been developed.

[0003] The PROTAC technology degrades target proteins through the ubiquitin-proteasome system (UPS), has an "event-driven" characteristic, does not require continuous occupation of the target, and can target traditional undruggable proteins. However, traditional PROTAC molecules still have many problems. First, small molecule PROTACs have a hook effect: at high concentrations, they tend to bind to a single E3 ligase or the target protein rather than binding to both proteins simultaneously, thus unable to play the function of pulling the target protein and the E3 ligase closer. This non-dose-dependent protein degradation greatly limits the clinical translation of PROTAC molecules. Second, the toxicity caused by the distribution of potential off-target effects in non-target normal tissues and organs also limits the clinical translation of PROTAC molecules. In addition, the spatial distance differences between different target proteins and E3 ligases require customized linkers, with high development costs and long cycles.

[0004] In CN114957231A, a GPX4 protein-targeting degradation chimera was developed based on the GPX4 inhibitor ML-162 and the E3 ubiquitin ligase CRBN protein ligand, which can bind to the GPX4 protein and trigger effective degradation, can significantly down-regulate the level of the GPX4 protein, and promote the generation of lipid droplets, causing ferroptosis in cells. However, this GPX4 protein-targeting degradation chimera still cannot solve the problems of the hook effect and off-target toxicity of PROTAC molecules.

[0005] In summary, how to develop a GPX4 protein degrader with good binding ability that can overcome the hook effect and off-target toxicity has become one of the technical problems to be urgently solved at present. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a GPX4 protein-targeted degrader and its preparation method and application. The GPX4 protein-targeted degrader can efficiently degrade the target protein GPX4 in the high-concentration reactive oxygen species (ROS) environment in tumor cells and induce ferroptosis of tumor cells.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a GPX4 protein-targeted degrader, which includes a tumor cell targeting unit, a ROS-responsive unit, an E3 ligase recognition unit, a self-assembly unit, and a GPX4 targeting and binding unit connected in sequence.

[0009] The tumor cell targeting unit includes a ligand of epidermal growth factor receptor.

[0010] In the present invention, the GPX4 protein-targeted degrader can form a barrel-shaped hexameric nanocarrier with highly ordered structure through dynamic self-assembly of polypeptide molecules in media such as water and PBS. It targets the epidermal growth factor receptor through the tumor cell targeting unit to achieve specific internalization of tumor cells. In the high-concentration reactive oxygen species (ROS) environment in tumor cells, the degrader breaks at the ROS-responsive unit, releasing the E3 ligase recognition unit, the self-assembly unit, and the GPX4 targeting and binding unit (named the released peptide segment), thereby further exposing the target head of the E3 ligase recognition unit. The self-assembly unit is connected to the E3 ligase recognition unit and the GPX4 targeting and binding unit at both ends, thus shortening the distance between the E3 ligase and GPX4. As Figure 1 shown, the released peptide segment can target both the E3 ligase and GPX4, and then effectively degrade the GPX4 protein.

[0011] The GPX4 protein-targeted degrader forms a β-barrel-shaped hexameric nanocarrier with highly ordered structure through dynamic self-assembly of polypeptide molecules. As the concentration of the GPX4 protein-targeted degrader increases, the size of the formed barrel-shaped hexameric nanocarrier increases and the surface area expands, providing sites for forming more stable GPX4-GPX4 protein-targeted degrader-E3 ligase ternary complexes, determining that the GPX4 protein-targeted degrader can achieve concentration-dependent and long-lasting degradation of the GPX4 protein, further improving the degradation efficiency of the GPX4 protein and inducing ferroptosis of tumor cells.

[0012] Preferably, the ligand of the epidermal growth factor receptor includes an EGFR ligand, and the amino acid sequence of the EGFR ligand includes the sequence shown in SEQ ID NO:1. The specific sequence of SEQ ID NO:1 is: LARLLT.

[0013] In the present invention, the sequence shown in SEQ ID NO:1 can target the epidermal growth factor receptor, enabling the GPX4 protein targeting degrader to enter epidermal growth factor-positive tumor cells and exert specific degradation of the GPX4 protein within the tumor cells. The high selectivity of the EGFR ligand and the reversible coupling strategy with the E3 ligase recognition unit can achieve dual-target synergistic enhancement.

[0014] Preferably, the ROS-responsive unit comprises 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butyric acid (4-((2-((2-((2-aminoethyl)thio)propan-2-yl)thio)ethyl)amino)-4-oxobutanoic acid), and the structure of 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butyric acid is shown in Formula I:

[0015]

[0016] In the present invention, 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butyric acid is utilized as the ROS-responsive linker bridge. On the one hand, the ROS-responsive unit is used to connect the tumor cell targeting unit and the releasing peptide segment, improving the safety and targeting of the degrader. On the other hand, after the GPX4 protein targeting degrader enters the tumor cell microenvironment, it triggers the cleavage of the ROS-responsive unit, converting the GPX4 protein targeting degrader from a prodrug into an active form, and "masking" the E3 ligase ligand in the β-barrel through steric hindrance effect, achieving tumor activation of the degrader.

[0017] Preferably, the E3 ligase recognition unit comprises a VHL protein ligand.

[0018] Preferably, the amino acid sequence of the VHL protein ligand comprises the sequence shown in SEQ ID NO:2, and the specific sequence of SEQ ID NO:2 is: ALAPYIP.

[0019] Preferably, the self-assembly unit comprises a polypeptide with self-assembly driving ability, and the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO:3, and the specific sequence of SEQ ID NO:3 is: IHIHIYIGPGIHIHIYI.

[0020] The self-assembly unit of the present invention creatively selects the sequence shown in SEQ ID NO: 3, initiates self-assembly through β-sheet, and realizes spatial ordered arrangement and multivalent binding interface through the regulation of the turn of the GPG amino acid fragment in the center of the self-assembly unit, greatly improving the formation of hexamers of the β-barrel structure and well overcoming the "hook effect" existing in the PROTAC technology.

[0021] Preferably, the GPX4 targeting binding unit includes a GPX4 targeting binding protein, and the amino acid sequence of the GPX4 targeting binding protein includes the sequence shown in SEQ ID NO: 4, and the specific sequence of SEQ ID NO: 4 is: CRAWYQNYCALRR.

[0022] Preferably, the structure of the GPX4 protein targeting degrader is as shown in Formula II:

[0023]

[0024] The schematic structural diagram of the GPX4 protein targeting degrader with the structure shown in Formula II is as Figure 2 shown. The ROS-responsive unit breaks at the disulfide bond, releasing the E3 ligase recognition unit, the self-assembly unit and the GPX4 targeting binding unit, thereby further realizing the targeting effect on GPX4 and the E3 ligase.

[0025] In the second aspect, the present invention provides a preparation method of the GPX4 protein targeting degrader as described in the first aspect, and the preparation method includes the following steps:

[0026] Using the tumor cell targeting unit, the ROS-responsive unit, the E3 ligase recognition unit, the self-assembly unit and the GPX4 targeting binding unit as raw materials, the GPX4 protein targeting degrader is synthesized by solid-phase synthesis.

[0027] Preferably, the preparation method includes the following steps:

[0028] (1) Fix the C-terminus of the first amino acid of the GPX4 targeting binding unit on the resin, and protect the N-terminus with Fmoc.

[0029] (2) Remove the N-terminal protection of the first amino acid of the GPX4 targeting binding unit in the deprotection solution, perform deprotection detection with a detection reagent, add the Fmoc-pretreated amino acid to the resin from which the protection has been removed for reaction, and sequentially connect the GPX4 targeting binding unit, the self-assembly unit and the E3 ligase recognition unit to connect the amino acids into a polypeptide fixed on the resin.

[0030] (3) Perform an amide condensation reaction on the polypeptide obtained in step (2) and the ROS-responsive unit.

[0031] (4) The product obtained in step (3) is successively combined with the amino acids of the tumor cell targeting unit, the product is removed from the resin, and the GPX4 protein targeting degrader is obtained through cleavage and purification.

[0032] Preferably, the resin described in step (1) includes Wang resin.

[0033] Preferably, the deprotection solution described in step (2) includes a dimethylformamide (DMF) solution containing piperidine.

[0034] Preferably, the volume fraction of piperidine in the deprotection solution is 10 - 30%, for example, it can be 10%, 15%, 20%, 25%, 30%, etc.

[0035] Preferably, the detection reagent described in step (2) includes ninhydrin test solution.

[0036] In a third aspect, the present invention provides an application of the GPX4 protein targeting degrader as described in the first aspect in the preparation of a drug for treating epidermal growth factor - positive tumors.

[0037] Preferably, the epidermal growth factor - positive tumors include any one or a combination of at least two of bladder cancer, cervical cancer, or neuroblastoma.

[0038] The numerical ranges described in the present invention not only include the above - listed point values, but also include any point values between the above - mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the ranges.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention designs a GPX4 protein targeting degrader based on a hexamer self - assembly linker, and improves its selectivity by designing a "double - gated" prodrug system. Its core design is as follows: through the dynamic self - assembly of polypeptide molecules, a β - barrel - shaped hexameric nanocarrier with highly ordered structure is formed, and the surface is modified with a tumor cell targeting unit to achieve specific internalization of tumor cells; under the triggering of the high reactive oxygen species (ROS) microenvironment in tumor cells, the chemical bond of the ROS response unit is broken, and the exposed E3 ligase recognition unit then recruits the E3 ligase complex and the GPX4 protein simultaneously, activating the ubiquitin - proteasome pathway (UPS) to achieve efficient and specific degradation of the GPX4 protein.

[0041] The β - barrel - shaped hexameric nanocarrier self - assembled by the GPX4 protein targeting degrader has a stable structure, and the formed β - barrel - shaped hexameric nanocarrier greatly increases the binding sites of the GPX4 protein and the E3 ligase, further improving the degradation efficiency of the GPX4 protein and inducing ferroptosis in tumor cells. Brief Description of the Drawings

[0042] Figure 1 It is a schematic diagram of the action of the GPX4 protein-targeted degrader in tumor cells.

[0043] Figure 2 It is a schematic diagram of the structure of the GPX4 protein-targeted degrader.

[0044] Figure 3 It is a synthetic route diagram of the ROS-responsive unit in Example 1.

[0045] Figure 4 It is a test result diagram of the time dependence of the GPX4 protein-targeted degrader in Test Example 1.

[0046] Figure 5 It is a test result diagram of the concentration dependence of the GPX4 protein-targeted degrader in Test Example 2.

[0047] Figure 6 It is a test result diagram of the inhibitory effect of the GPX4 protein-targeted degrader on tumor growth in Test Example 3. Detailed Description of the Invention

[0048] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0049] Example 1

[0050] This example provides a GPX4 protein-targeted degrader, the structural formula of which is shown in Formula II and is synthesized by the solid-phase peptide synthesis method.

[0051]

[0052] The specific method of the solid-phase peptide synthesis method is as follows:

[0053] (1) Experimental instruments and materials:

[0054] Dimethylformamide (DMF), piperidine, Wang resin, dichloromethane (DCM), ninhydrin reaction reagent (ninhydrin, vitamin C and phenol), benzotriazol - N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, Fmoc-alanine (Fmoc-Ala-OH), Fmoc-leucine (Fmoc-Leu-OH), Fmoc-proline (Fmoc-Ile-OH), Fmoc-tyrosine (Fmoc-Tyr(Trt)-OH), Fmoc-isoleucine (Fmoc-Gln(Trt)-OH), Fmoc-histidine (Fmoc-His(Trt)-OH), Fmoc-glycine (Fmoc-Gly-OH), Fmoc-cysteine (Fmoc-Cys(Trt)-OH), Fmoc-arginine (Fmoc-Arg(Pbf)-OH), Fmoc-threonine (Fmoc-Thr(Tos)-OH), acid anhydride, solid-phase peptide synthesis tube, etc.

[0055] (2) Preparation of experimental solutions:

[0056] Deprotection solution: Mix hexahydropyridine and DMF according to a volume ratio of 1:4;

[0057] Reaction solution: Mix NMM and DMF according to a volume ratio of 1:24;

[0058] Cleavage solution: Mix TFA, TIS and H2O, and the volume fractions of each component in the mixed solution are: 92.5% TFA, 2.5% TIS and 2.5% H2O;

[0059] Ninhydrin test solution: One drop each of ninhydrin, vitamin C and phenol.

[0060] (3) Synthesis method:

[0061] (3.1) Synthesize the E3 ligase recognition unit, self-assembly unit and GPX4 targeting binding unit, and its amino acid sequence is the sequence shown in SEQ ID NO:5. The specific sequence of SEQ ID NO:5 is: ALAPYIPIHIHIYIGPGIHIHIYICRAWYQNYCALRR. The sequence shown in SEQ ID NO:5 is connected to the resin in sequence starting from arginine (R).

[0062] (3.1.1) Resin swelling and Fmoc deprotection:

[0063] Weigh 0.1 g of Wang resin and put it into a solid-phase peptide synthesis tube. Add DMF to swell it for 30 min. Remove the DMF by suction, and perform the Fmoc deprotection reaction with the deprotection solution. Place it on a shaker for 10 min. Remove the deprotection solution by suction, and wash it 3 times with DMF and DCM. Take 10 mg of Wang resin from the solid-phase peptide synthesis tube and put it into a test tube. Wash it 2 times with ethanol. After the ninhydrin test shows a dark blue color, which is a positive result, prepare to introduce the first amino acid (arginine) and enter the amino acid condensation reaction.

[0064] (3.1.2) Amino acid condensation:

[0065] Take 10-fold equivalents of Fmoc-arginine and HBTU, dissolve them in 7 mL of the reaction solution, put them into the solid-phase peptide synthesis tube, and stir for the reaction. After 1 h, take 10 mg of Wang resin from the solid-phase peptide synthesis tube and put it into a test tube. Wash it 2 times with ethanol. After the ninhydrin test shows no color change, which is a negative result, it proves that the condensation reaction is successful. Remove the liquid in the solid-phase peptide synthesis tube by suction, and wash it 3 times with DMF and DCM respectively to obtain the peptide resin after the condensation of the first amino acid.

[0066] (3.1.3) Repeat the "Fmoc deprotection - amino acid condensation" steps of (3.1.1) and (3.1.2) for the peptide resin obtained in step (3.1.2) until the reaction of the last amino acid Fmoc-alanine (A) in the sequence shown in SEQ ID NO:5 is completed.

[0067] (3.2) Connect the ROS-responsive unit:

[0068] (3.2.1) First, add 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-amine) to the acid anhydride for a coupling reaction to obtain the ROS-responsive unit 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butanoic acid, and its reaction process is as Figure 3 shown.

[0069] (3.2.2) Condense the sequence shown in SEQ ID NO:5 and 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butanoic acid through amide condensation. The product of the amide condensation is deprotected and purified to obtain the structure shown in formula Ⅲ:

[0070]

[0071] (3.3) Connect the tumor cell targeting unit and product verification:

[0072] The product obtained in step (3.2) was successively linked with T-L-L-R-A-L (Fmoc-amino acid) of the tumor cell targeting unit, and deprotection was carried out after the last amino acid was completed, and ninhydrin was used to verify that the deprotection was complete.

[0073] After the reaction was completed, the resin was washed 3 times with DMF and DCM respectively, washed 2 times with methanol, and then dried by suction for 20 min. The synthesized peptide resin was taken out from the solid-phase peptide synthesis tube, lysed in the lysis solution at 25 °C for 2 h, and the lysis solution was ice-bathed for 20 min first. After filtering the resin, it was evaporated to dryness on a rotary evaporator and washed 3 times with anhydrous ether under ice-bath conditions to obtain the crude peptide.

[0074] The crude peptide was purified by preparative reverse-phase HPLC, and the purity was detected by HPLC > 95%. The obtained pure peptide was identified by mass spectrometry, and the measured molecular weight result was the same as that of the target molecule (the structure shown in formula II).

[0075] Test Example 1

[0076] In this test example, a time-dependent test was carried out on the GPX4 protein-targeted degrader obtained in Example 1.

[0077] The human bladder cancer EJ cell line was selected. The cells were inoculated in a six-well plate. When they grew to 90%, the medium was removed, the cells were washed with PBS, and the GPX4 protein-targeted degrader obtained in Example 1 was added to a final concentration of 10 μM. The cells were placed in an incubator at 37 °C and 5% CO2. Cells were collected at 12, 24, 36, 48, and 72 hours respectively. The cells were lysed with lysis buffer, the supernatant was taken by centrifugation, the proteins were separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, incubated with the primary antibody and secondary antibody against GPX4, and finally Tubulin was used as an internal reference control, and the imaging reagent was used for imaging and observation.

[0078] By comparing the intensities of the GPX4 protein bands at different time points, the degradation effect of the GPX4 protein-targeted degrader on the GPX4 protein was evaluated. The results are as Figure 4 shown. It can be seen that under the action of the 10 μM GPX4 protein-targeted degrader at different times in EJ cells, the expression level of the GPX4 protein decreased in a time-dependent manner, thus verifying that the GPX4 protein-targeted degrader has a time-dependent protein degradation effect.

[0079] Test Example 2

[0080] In this test example, a concentration-dependent test was carried out on the GPX4 protein-targeted degrader obtained in Example 1.

[0081] Refer to the method of Test Example 1 to test the concentration dependence of the GPX4 protein-targeting degrader. Seed the same EJ cells as in Test Example 1 into a six-well plate. When they grow to 90%, remove the culture medium, wash the cells with PBS, and add the GPX4 protein-targeting degrader obtained in Example 1 for concentration-dependent incubation:

[0082] The concentrations of the GPX4 protein-targeting degrader are 2 μM, 5 μM, 10 μM, and 20 μM respectively. Incubate the GPX4 protein-targeting degrader with EJ cells for 5 h. After each of the above incubations is completed, refer to the method of Test Example 1 to verify the expression level of the GPX4 protein.

[0083] The results are as Figure 5 shown. It can be seen that under the action of different concentrations (2 μM, 5 μM, 10 μM, 20 μM) of the GPX4 protein-targeting degrader, the expression level of the GPX4 protein in EJ cells shows a concentration-dependent decrease, thus verifying that the GPX4 protein-targeting degrader has a concentration-dependent protein degradation effect.

[0084] Test Example 3

[0085] This test example tests the inhibitory effect of the GPX4 protein-targeting degrader on tumor growth.

[0086] (1) Construction of a tumor mouse model:

[0087] By subcutaneously injecting 100 μL of 5×10 7 cells / mL of EJ cells into female Balb / c nude mice (6 - 8 weeks old, 18 - 20 g, purchased from Beijing Spearf Bio-Technology Co., Ltd.) once, a tumor model was established.

[0088] (2) Experimental grouping and intervention method:

[0089] When the tumor volume reaches approximately 100 mm 3 ³, randomly divide the mice into two groups (6 mice in each group), and inject PBS and the GPX4 protein-targeting degrader obtained in Example 1 via the tail vein respectively. Inject the drug once every 3 days for 6 consecutive times. The dose is set at 8 mg / kg, and the injection volume is 100 μL / mouse.

[0090] During the drug administration treatment, measure the three dimensions (length, width, height) of the tumors of the mice with a caliper every 2 days, and calculate the tumor volume. Compare the changes in tumor volume between the two groups during the treatment. The results are as Figure 6 shown.

[0091] It can be seen that the tumor volume of the mice injected with PBS increased significantly with the extension of time, and the tumor growth rate was fast. In contrast, the tumor growth rate of the mice injected with the GPX4 protein targeting degrader decreased significantly, indicating that the GPX4 protein targeting degrader obtained in Example 1 has excellent anti-tumor effects.

[0092] Based on the data analysis of the above test examples, it can be known that the GPX4 protein targeting degrader provided by the present invention can be activated in tumor cells through a high-concentration ROS environment, can recruit multiple GPX4 proteins and E3 ligases simultaneously, promote the formation of a stable multivalent complex, and effectively mediate the ubiquitination and proteasomal degradation of GPX4. With the increase in the concentration of the GPX4 protein targeting degrader, the size of the formed barrel-shaped hexameric nanocarrier increases, and the surface area expands, providing sites for the formation of more stable GPX4-GPX4 targeting degrader-E3 ligase ternary complexes, thereby realizing concentration-dependent protein degradation. In addition, due to the long-term retention characteristics of the β-barrel hexameric nanocarrier in cells, the GPX4 protein targeting degrader of the present invention can achieve continuous GPX4 protein degradation in cells.

[0093] In summary, the GPX4 protein targeting degrader provided by the present invention shows good GPX4 protein degradation effects and realizes significant tumor growth inhibition in animal models, verifying its great potential and application value as a novel anti-cancer therapeutic agent.

[0094] The applicant declares that the present invention uses the above examples to illustrate the technical solutions of the present invention, but the present invention is not limited to the above examples, that is, it does not mean that the present invention must rely on the above examples to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and disclosure scope of the present invention.

[0095] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0096] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate way. To avoid unnecessary repetition, the present invention will not describe various possible combination methods separately.

Claims

1. A GPX4 protein-targeted degrader, characterized in that, The GPX4 protein-targeted degrader comprises a tumor cell targeting unit, a ROS-responsive unit, an E3 ligase recognition unit, a self-assembly unit, and a GPX4-targeted binding unit which are connected in sequence; The tumor cell targeting unit comprises a ligand of epidermal growth factor receptor.

2. The GPX4 protein-targeting degrader according to claim 1, wherein The ligand of epidermal growth factor receptor comprises an EGFR ligand, and the amino acid sequence of the EGFR ligand comprises the sequence shown in SEQ ID NO:

1.

3. The GPX4 protein-targeting degrader according to claim 1 or 2, wherein The ROS-responsive unit comprises 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butyric acid, and the structure of 4-oxo-4-[(2-{[2-({2-aminoethyl}thio)propan-2-yl]thio}ethyl)amino]butyric acid is shown in Formula I:

4. The GPX4 protein-targeting degrader according to any one of claims 1-3, characterized in that, The E3 ligase recognition unit comprises a VHL protein ligand; Preferably, the amino acid sequence of the VHL protein ligand comprises the sequence shown in SEQ ID NO:

2.

5. The GPX4 protein-targeting degrader according to any one of claims 1-4, characterized in that, The self-assembly unit comprises a polypeptide with self-assembly driving ability, and the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO:

3.

6. The GPX4 protein-targeted degrader according to any one of claims 1-5, characterized in that, The GPX4-targeted binding unit comprises a GPX4-targeted binding protein, and the amino acid sequence of the GPX4-targeted binding protein comprises the sequence shown in SEQ ID NO:

4.

7. The GPX4 protein-targeting degrader according to any one of claims 1-6, characterized in that, The structure of the GPX4 protein-targeted degrader is shown in Formula II:

8. A method for preparing a GPX4 protein-targeted degrader according to any one of claims 1-7, characterized in that, The preparation method comprises the following steps: Using the tumor cell targeting unit, the ROS-responsive unit, the E3 ligase recognition unit, the self-assembly unit, and the GPX4-targeted binding unit as raw materials, the GPX4 protein-targeted degrader is synthesized by solid-phase synthesis.

9. The preparation method of the GPX4 protein-targeting degrader according to claim 8, wherein, The preparation method comprises the following steps: (1) Fix the C-terminus of the first amino acid of the GPX4-targeted binding unit on the resin, and protect the N-terminus with Fmoc; (2) Remove the N-terminal protection of the first amino acid of the GPX4-targeted binding unit in the deprotection solution, perform deprotection detection with a detection reagent, add the Fmoc-pretreated amino acid to the deprotected resin for reaction, and sequentially connect the GPX4-targeted binding unit, the self-assembly unit, and the E3 ligase recognition unit to connect the amino acids into a polypeptide fixed on the resin; (3) Perform an amide condensation reaction on the polypeptide obtained in step (2) and the ROS-responsive unit; (4) Combine the product obtained in step (3) with the amino acids of the tumor cell targeting unit in sequence, remove the product from the resin, and obtain the GPX4 protein-targeted degrader through cleavage and purification.

10. Use of a GPX4 protein-targeted degrader according to any one of claims 1-7 in the preparation of a drug for treating epidermal growth factor-positive tumors; Preferably, the epidermal growth factor-positive tumors include any one or a combination of at least two of bladder cancer, cervical cancer, or neuroblastoma.

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