Cell-penetrating peptide modified enzyme-sensitive PDC-type PROTAC and preparation method and application thereof

Abstract

The application discloses a cell-penetrating peptide modified enzyme-sensitive PDC type PROTAC as well as a preparation method and application thereof, and belongs to the technical field of tumor targeted therapy. The cell-penetrating peptide modified enzyme-sensitive PDC type PROTAC is obtained by modifying a cell-penetrating peptide and an enzyme-sensitive linker (GFLG) to a PROTAC of an anti-tumor drug target protein ligand, has the ability to release the PDC type PROTAC under catalysis of cathepsin B, has a smaller influence on cell viability of U251 cells, U87 cells and HEK293 cells, has proliferation inhibition activity on the U251 cells and the U87 cells, can degrade target proteins in the U251 cells and the U87 cells, can induce apoptosis of the U251 cells and the U87 cells, has an influence on U251 cell cycles, can be used for preparing an anti-tumor drug (target protein degradation and membrane penetration), has a good application prospect in preparation of a drug for targeting human brain glioma cells, and can be used as another important field for PROTAC drug development.

Description

Technical Field

[0001] This invention belongs to the field of tumor targeted therapy technology, specifically relating to enzyme-sensitive PDC-type PROTAC modified with cell membrane peptides, its preparation method, and its application. Background Technology

[0002] Cancer, also known as malignant tumor, is a disease caused by the abnormal control of cell division and proliferation. It occurs when a cell in a local tissue loses normal genetic regulation of its growth under the influence of various carcinogenic factors, leading to its clonal abnormal proliferation and the formation of a new growth. This new growth often manifests as a mass in a local tissue, and cancer cells can also metastasize to other parts of the body via lymphatic vessels or blood vessels, forming new metastatic lesions. In recent years, the incidence of new cancer cases has been increasing year by year, causing various impacts on society, including economic burden, depletion of medical resources, and loss of labor. Therefore, the prevention, early diagnosis, and treatment of cancer are important issues in the field of global public health.

[0003] Proteolysis-targeting chimeras (PROTACs) are an emerging drug discovery technology that utilizes the intracellular ubiquitin-proteasome system (UPS) to degrade specific proteins. A PROTAC molecule typically consists of three parts: a target protein ligand, an E3 ubiquitin ligase ligand, and a linker connecting the two ligands. By bringing the target protein and the E3 ubiquitin ligase close together, PROTACs can label the target protein as a protein to be degraded, thereby enabling its degradation via the UPS system. When the target protein ligand is an anti-tumor drug, PROTACs can target and degrade a variety of proteins closely related to tumorigenesis and development, such as nuclear receptor proteins (e.g., androgen receptor AR and estrogen receptor ER), brominated structures and superterminal protein families (BET), and protein kinases. The degradation of these proteins can inhibit tumor cell proliferation and migration, and promote their senescence and apoptosis. Although PROTAC technology has shown great potential in the field of cancer treatment, its large molecular weight and surface area typically lead to poor solubility and membrane permeability when used to treat tumors. Furthermore, the design of PROTAC molecules is challenging and carries the potential for off-target effects, limiting its widespread clinical application. Therefore, further optimization of PROTAC molecule design is essential.

[0004] Peptide-drug conjugates (PDCs) are an emerging type of targeted therapy that combines the high tissue penetration of peptides with the potent therapeutic effects of drugs. PDCs typically consist of three key components: a targeting peptide (or homing peptide), a linker, and a cytotoxic drug (or payload). Despite the numerous advantages of PDCs, their development also faces several challenges. For example, peptides exhibit poor stability in vivo and are easily degraded by proteases; the synthesis and purification of PDCs are relatively complex, requiring high-precision technologies and equipment. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide cell membrane-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, its preparation method and application, and solve the problem of low membrane permeability in the process of PROTAC treatment of cells.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The first aspect of the present invention discloses an enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides, which is obtained by modifying PROTAC with cell-penetrating peptides;

[0008] The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.10 or SEQ ID NO.11, and the target protein ligand in the PROTAC is an anti-tumor drug.

[0009] Preferably, the amino acid sequence of PROTAC is shown in SEQ ID NO.1.

[0010] More preferably, it also includes an enzyme-sensitive linker, a cell-penetrating peptide, and an enzyme-sensitive linker co-modifying PROTAC;

[0011] The amino acid sequence of the enzyme-sensitive linker is GFLG.

[0012] More preferably, when the cell-penetrating peptide is used alone for modification, the cell-penetrating peptide and PROTAC are linked by a covalent bond; when the cell-penetrating peptide and enzyme-sensitive linker are used together for modification, the cell-penetrating peptide, enzyme-sensitive linker and PROTAC are linked by a covalent bond.

[0013] In a second aspect, the present invention discloses a method for synthesizing the above-mentioned cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, wherein the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC is prepared by an artificial solid-phase synthesis method based on FMOC amino acids.

[0014] A third aspect of the present invention discloses the application of the above-mentioned cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC in the preparation of antitumor drug formulations.

[0015] Preferably, the antitumor drug is an anti-solid tumor drug.

[0016] Preferably, the antitumor drug is an antiglioma drug.

[0017] More preferably, the anti-glioma drug is a drug that targets human brain glioma cells.

[0018] Preferably, the concentration of the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC is 1.0 × 10⁻⁶. -1 ~100.0 μmol / L.

[0019] A fourth aspect of the present invention discloses an anti-glioma composition, characterized in that it comprises other active ingredients having anti-glioma activity, the above-mentioned cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, and one or more pharmaceutically acceptable carriers or excipients.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides an enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide. 1) A combined PDC and PROTAC design strategy, using a cell-penetrating peptide to modify a PROTAC with anti-tumor capabilities, significantly improves the PROTAC's anti-tumor cell proliferation activity, protein degradation effect, and pro-apoptotic ability, resulting in high efficacy and reduced dosage of PDC-type PROTAC. 2) When using the cell-penetrating peptide with the amino acid sequence shown in SEQ ID NO. 10, the modified PROTAC exhibits better release than the unmodified PROTAC, indicating better membrane permeability. 3) When using the cell-penetrating peptide with the amino acid sequence shown in SEQ ID NO. 11, the modified PROTAC shows better targeting and membrane penetration effects on tumor blood vessels and tumor cells compared to the unmodified PROTAC. 4) The modified PROTAC exhibits a certain degree of safety at the cellular level. 5) Compared to the unmodified PROTAC, the modified PROTAC significantly enhances tumor proliferation inhibition activity and improves target protein degradation, demonstrating the rationality of the design strategy and addressing the off-target effects of traditional PROTACs. Therefore, this cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC can be used to prepare anti-tumor drugs and has good application prospects in the preparation of drugs targeting human glioma cells. It can be regarded as another important area of ​​PROTAC drug development.

[0022] Furthermore, co-modification of PROTAC with cell-penetrating peptides and enzyme-sensitive linkers yields superior results compared to modification with cell-penetrating peptides alone. Specifically: 1) The enzyme-sensitive linker has the ability to release PROTAC under the catalysis of cathepsin B, simultaneously achieving cell-penetrating and protein degradation effects; 2) It has minimal impact on the cell viability of U251, U87, and HEK293 cells; 3) It exhibits proliferative inhibitory activity against U251 and U87 cells, with significantly superior activity compared to PDC-type PROTAC; 4) It can degrade target proteins in U251 and U87 cells, induce apoptosis in both cells, and influence the U251 cell cycle. Therefore, this enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides can be used to prepare anti-tumor drugs (especially targeting protein degradation and permeabilization), showing promising application prospects in the preparation of drugs targeting human glioma cells, and representing another important area in PROTAC drug development.

[0023] The method for preparing cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC provided by this invention is prepared by chemical synthesis. The technology is mature, easy to prepare in large quantities, and has good stability. It has potential application prospects for the development of the currently limited number of PDC-type PROTACs. Attached Figure Description

[0024] Figure 1 A flowchart of the synthesis method for enzyme-sensitive PDC-type PROTAC modified with cell membrane-penetrating peptides;

[0025] Figure 2 A flowchart of the synthesis method of enzyme-sensitive PDC-type PROTAC modified with targeted transmembrane peptides for tumor blood vessels and tumor cells;

[0026] Figure 3 A diagram illustrating the release process of PROTAC in enzyme-sensitive PDC-type PROTAC modified with cell membrane-penetrating peptides (under enzyme presence conditions);

[0027] Figure 4 A diagram illustrating the linker breakage process in the enzyme-sensitive PDC-type PROTAC control modified with cell-penetrating peptides (under enzyme presence conditions).

[0028] Figure 5 A diagram illustrating the release process of PROTAC in enzyme-sensitive PDC-type PROTAC modified with cell membrane-penetrating peptides (under conditions where the enzyme is absent);

[0029] Figure 6 A diagram illustrating the release process of PROTAC in enzyme-sensitive PDC-type PROTAC modified with peptides that target tumor blood vessels and tumor cells (under enzyme presence).

[0030] Figure 7 Figure showing the effect of PDC-type PROTAC modified with cell-penetrating peptides on the viability of HEK293 cells;

[0031] Figure 8 Figure showing the effect of PDC-type PROTAC modified with tumor blood vessels and tumor cell-targeting membrane-penetrating peptides on the viability of HEK293 cells;

[0032] Figure 9 Figure showing the inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptides against the proliferation of U251 cells;

[0033] Figure 10 The figure shows the inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptides against the proliferation of U87 cells.

[0034] Figure 11 Figure showing the inhibitory activity of PDC-type PROTAC modified with tumor blood vessel and tumor cell-targeting membrane-penetrating peptides on the proliferation of U251 cells;

[0035] Figure 12 Figure showing the inhibitory activity of PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting membrane-penetrating peptides on the proliferation of U87 cells;

[0036] Figure 13 The results of the degradation of intracellular target proteins by PDC-type PROTAC modified with cell-penetrating peptides are shown in the figure. Among them: (A) shows the degradation effect of HIF-S on intracellular target proteins in U251 cells, (C) shows the degradation effect of CPP-HIF-S on intracellular target proteins in U251 cells, (E) shows the degradation effect of CPP-GFLG-HIF-S on intracellular target proteins in U251 cells, and (B), (D) and (F) are semi-quantitative results of target protein expression levels in (A), (C) and (E), respectively.

[0037] Figure 14 The results of the degradation of intracellular target proteins by PDC-type PROTAC modified with cell-penetrating peptides are shown in the figure; where: (A) is the result of the degradation of intracellular target proteins by HIF-S, (C) is the result of the degradation of intracellular target proteins by CPP-HIF-S, (E) is the result of the degradation of intracellular target proteins by CPP-GFLG-HIF-S, and (B), (D) and (F) are semi-quantitative results of target protein expression levels in (A), (C) and (E), respectively;

[0038] Figure 15The results show the degradation effects of PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides on intracellular target proteins in U251 cells; where: (A) shows the degradation effect of Cyclo-A7R-RRR-HIF-S on intracellular target proteins in U251 cells, (C) shows the degradation effect of Cyclo-A7R-RRR-GFLG-HIF-S on intracellular target proteins in U251 cells, and (B) and (D) are semi-quantitative results of target protein expression levels in (A) and (C);

[0039] Figure 16 The results show the degradation effects of PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides on target proteins in U87 cells; where: (A) shows the degradation effect of Cyclo-A7R-RRR-HIF-S on target proteins in U87 cells, (C) shows the degradation effect of Cyclo-A7R-RRR-GFLG-HIF-S on target proteins in U87 cells, and (B) and (D) are semi-quantitative results of target protein expression levels in (A) and (C);

[0040] Figure 17 The effect of PDC-type PROTAC modified with cell-penetrating peptide on apoptosis of U251 cells is shown in the figure; where: (A) is the effect of CPP-HIF-S on apoptosis of U251 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0041] Figure 18 The effect of cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC on U251 cell apoptosis is shown in the figure; where: (A) shows the effect of CPP-GFLG-HIF-S on U251 cell apoptosis, and (B) shows the quantitative results of apoptotic cells in (A);

[0042] Figure 19 The effect of cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC on apoptosis of U87 cells is shown in the figure; where: (A) is the effect of CPP-GFLG-HIF-S on apoptosis of U87 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0043] Figure 20 The effect of PDC-type PROTAC modified with tumor blood vessels and tumor cell-targeting membrane-penetrating peptides on U251 cell apoptosis is shown in the figure; where: (A) shows the effect of Cyclo-A7R-RRR-HIF-S on U251 cell apoptosis, and (B) shows the quantitative results of apoptotic cells in (A);

[0044] Figure 21The effect of enzyme-sensitive PDC-type PROTAC modified with tumor blood vessels and tumor cells targeting membrane-penetrating peptides on apoptosis of U251 cells is shown in the figure; where: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on apoptosis of U251 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0045] Figure 22 The effect of PDC-type PROTAC on apoptosis of U87 cells is shown in the figure; where: (A) shows the effect of HIF-S on apoptosis of U251 cells, and (B) shows the quantitative results of apoptotic cells in (A);

[0046] Figure 23 The effect of enzyme-sensitive PDC-type PROTAC modified with tumor blood vessels and tumor cells targeting membrane-penetrating peptides on apoptosis of U87 cells is shown in the figure; where: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on apoptosis of U87 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0047] Figure 24 The effect of PDC-type PROTAC on the U251 cell cycle is shown in the figure; where: (A) is the effect of HIF-S on the U251 cell cycle, and (B) is the quantitative result of (A);

[0048] Figure 25 The effect of cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC on the cell cycle of U251 cells is shown in the figure; where: (A) is the effect of CPP-GFLG-HIF-S on the cell cycle of U251 cells, and (B) is the quantitative result of (A);

[0049] Figure 26 The effect of enzyme-sensitive PDC-type PROTAC modified with tumor blood vessels and tumor cells targeting membrane-penetrating peptides on the cell cycle of U251 cells is shown in the figure; where: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on the cell cycle of U251 cells, and (B) is the quantitative result of (A). Detailed Implementation

[0050] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0051] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0052] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0053] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0054] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0055] In this article, the amino acid sequence of the hypoxia-inducible factor-1α (HIF-1α) protein fragment is Leu-Ala-Pro(OH)-Tyr-Ile.

[0056] This invention provides a method for synthesizing PDC-type PROTAC modified with cell-penetrating peptides. The method involves linking a hypoxia-inducible factor 1α (HIF-1α) protein fragment (sequence shown as SEQ ID NO. 6 in Table 1) with sorafenib via mini-PEG to obtain PROTAC (sequence shown as SEQ ID NO. 1 in Table 1); then modifying the PROTAC with cell-penetrating peptides to obtain PDC-type PROTAC modified with cell-penetrating peptides.

[0057] Among them, the cell-penetrating peptide is CPP (sequence shown as SEQ ID NO.10 in Table 1) or Cyclo-A7R-RRR (sequence shown as SEQ ID NO.11 in Table 1).

[0058] Furthermore, PDC-type PROTACs can be modified by co-modifying cell-penetrating peptides and enzyme-sensitive linkers (GFLG) to obtain PDC-type PROTACs modified with cell-penetrating peptides.

[0059] Table 1 Amino acid sequence list

[0060]

[0061]

[0062] Note: In the amino acid sequence, "L" represents "Leucine", "A" represents "Alanine", "P(OH)" represents "hydroxy-L-proline", "Y" represents "Tyrosine", "I" represents "Isoleucine", "S" represents "Sorafenib", "C(...)" represents cyclization, "f" represents "D-phenylalanine", "Φ" represents "L-2-naphthylalanine", "R" represents "L-arginine", "r" represents "D-arginine", "E" represents "Glutamic Acid", "G" represents "Glycine", "F" represents "L-phenylalanine", "K" represents "Lysine", and "P" represents "Proline".

[0063] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0064] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage.

[0065] I. Experimental Materials

[0066] 1. Experimental drugs and reagents: Rink Amide-MBHA resin (Bailinwei), Fmoc-Arg(Pbf)-Wang resin (Bailinwei), Fmoc protected amino acids, piperidine (Sinopharm Group), DMF (Comeo Chemical Reagent), DCM (Comeo Chemical Reagent), HATU (Bide Pharmaceutical), HOBt (Bide Pharmaceutical), DIPEA (Bide Pharmaceutical), PyBop (Bide Pharmaceutical), TFA (Aladdin), TIS (Aladdin), 1,3-dimethoxybenzene (Aladdin), DODT (Aladdin), chromatographic acetonitrile (Aladdin), PBS buffer (CORNING), DMEM (CORNING), penicillin-streptomycin solution (double antibody) (MCE), fetal bovine serum (NEWZERUM), Trypsin (CORNING), DMSO (MP Biomedicals), MTT (Solarbio).

[0067] 2. Cells used in the experiment: U251 (human glioma cells), U87 (human glioma cells), and HEK293 (human embryonic kidney cells).

[0068] II. Synthetic cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC

[0069] like Figure 1 As shown, enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides was synthesized using a solid-phase synthesis method based on FMOC amino acids.

[0070] 1. Synthesis of sorafenib hydrolysis product (4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)pyridinecarboxylic acid)

[0071] Sorafenib (1.00 g, 2.15 mmol) and NaOH (1.40 g, 32.27 mmol) were dissolved in anhydrous ethanol and reacted at 80 °C under N2 protection for 10 hours. Excess ethanol was removed, and dilute hydrochloric acid (2 mol / L) was added to neutralize the excess alkali to pH 3. The system was filtered under reduced pressure to obtain a purple solid filter cake (0.93 g, 96.0% yield). MS (ESI, m / z): 450.00 [MH] - .

[0072] 2. Synthesis of CPP-HIF-S

[0073] 1) Synthesis of CPP membrane-penetrating peptides

[0074] Take 50.0 mg of Rink Amide-MBHA resin (0.32 mmol / g, 0.016 mmol) into a reaction vessel (centrifuge column), add 5 mL of DMF / DCM (1:1), allow to swell for 45 minutes, then filter under reduced pressure and discard the filtrate. Add 20% piperidine / DMF for 10 minutes to remove the FMOC protecting group, filter under reduced pressure, and repeat the above operation twice to completely remove the Fmoc protecting group at the N-terminus of the Rink Amide-MBHA resin. After filtration, thoroughly wash the Rink Amide-MBHA resin with DCM / DMF. Fmoc-Glu(O-2PhiPr)-OH (39.0 mg, 0.08 mmol), HATU (30.3 mg, 0.08 mol), HOBt (12.2 mg, 0.08 mmol), and DIPEA (37.6 μL, 0.16 mmol) were dissolved in an appropriate amount of DMF. The mixture was added to a centrifuge column and reacted for 45 minutes. The mixture was then filtered and the resin was thoroughly washed with DCM / DMF. The amino acids are sequentially linked according to the sequence shown in Table 1 as SEQ ID NO.2: Fmoc-Glu-OAll (32.8 mg, 0.08 mmol), Fmoc-D-Arg(Pbf)-OH (51.9 mg, 0.08 mmol), Fmoc-Arg(Pbf)-OH (51.9 mg, 0.08 mmol), Fmoc-D-Arg(Pbf)-OH (51.9 mg, 0.08 mmol), Fmoc-2-Nal-OH (35.0 mg, 0.08 mmol) and Fmoc-D-Phe-OH (31.0 mg, 0.08 mmol). Cyclization was performed using PyBop (41.6 mg, 0.08 mmol), HOBt (12.2 mg, 0.08 mmol), and DIPEA (37.6 μL, 0.16 mmol). Two cyclization reactions were performed, one for 3 hours and the other for 8 hours, to obtain the CPP transmembrane peptide.

[0075] 2) Synthesis of CPP-HIF-S

[0076] Following the sequence order shown in Table 1 as SEQ ID NO.2, the following amino acids were sequentially linked to the CPP transmembrane peptide: Fmoc-L-Ile-OH (28.3 mg, 0.08 mmol), Fmoc-L-Tyr(tBu)-OH (36.8 mg, 0.08 mmol), Fmoc-4-tert-butoxy-L-proline (32.8 mg, 0.08 mmol), Fmoc-Ala-OH (23.8 mg, 0.08 mmol), and Fmoc-Leu-OH (28.3 mg, 0.08 mmol) to obtain the HIF moiety. Then, mini-PEG (30.8 mg, 0.08 mmol) was added, and finally, the hydrolysis product of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to a centrifuge column under the action of N,N-diisopropylcarbodiimide (DIC; 17.9 μL, 0.08 mmol), and the reaction was carried out overnight to link sorafenib to the polypeptide chain. After synthesis, the product was cleaved from the resin using a cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H2O = 90:2.5:2.5:2.5:2.5). DCM was added to the centrifuge column to thoroughly wash the product and the liquid was collected. The solvent was evaporated, ice-cold ether was added to the centrifuge tube, and the product was centrifuged at 8000 rpm for 20 minutes. The supernatant was then discarded, ice-cold ether was added to the centrifuge tube again, the product was gently washed, and the product was centrifuged at 8000 rpm for 5 minutes. Discard the supernatant to obtain crude peptide CPP-HIF-S, and store it in a -80°C freezer.

[0077] 3) Purification

[0078] The crude peptide CPP-HIF-S was purified by linear gradient elution using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) on a Shimadzu LC-2030. Purity was analyzed using a Shimadzu LC-2030C 3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). Characterization was performed using a Hexin Instruments CMI-1600MALDI-TOF-MS. The purity of the purified peptide was >90.0% at 254 nm.

[0079] 3. Synthesis of CPP-GFLG-HIF-S

[0080] 1) Synthesis of CPP membrane-penetrating peptides

[0081] The synthesis method is the same as step 2.

[0082] 2) Synthesis of CPP-GFLG

[0083] Following the sequence order shown in SEQ ID NO.3 in Table 1, amino acids were sequentially linked to the CPP transmembrane peptide: Fmoc-Gly-OH (23.8 mg, 0.08 mmol), Fmoc-Leu-OH (28.3 mg, 0.08 mmol), Fmoc-Phe-OH (31.0 mg, 0.08 mmol), and Fmoc-Gly-OH (23.8 mg, 0.08 mmol) to obtain CPP-GFLG.

[0084] 3) Synthesis of CPP-GFLG-HIF-S

[0085] Following the sequence order shown in SEQ ID NO.3 in Table 1, the following amino acids were sequentially ligated onto CPP-GFLG: Fmoc-L-Ile-OH (28.3 mg, 0.08 mmol), Fmoc-L-Tyr(tBu)-OH (36.8 mg, 0.08 mmol), Fmoc-4-tert-butoxy-L-proline (32.8 mg, 0.08 mmol), Fmoc-Ala-OH (23.8 mg, 0.08 mmol), and Fmoc-Leu-OH (28.3 mg, 0.08 mmol) to obtain the HIF moiety. Then, mini-PEG (30.8 mg, 0.08 mmol) was ligated. Finally, the hydrolysis product of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to a centrifuge column under DIC (17.9 μL, 0.08 mmol) and reacted overnight to ligate sorafenib onto the polypeptide chain. After synthesis, the product was cleaved from the resin using a cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H2O = 90:2.5:2.5:2.5:2.5). DCM was added to a centrifuge column to thoroughly wash the product, and the liquid was collected. The solvent was evaporated, and ice-cold ether was added to the centrifuge tube. The product was centrifuged at 8000 rpm for 20 minutes, and the supernatant was discarded. Ice-cold ether was added again to the centrifuge tube, and the product was gently washed and centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded to obtain the crude peptide CPP-GFLG-HIF-S, which was stored at -80°C.

[0086] 4) Purification

[0087] The crude peptide CPP-GFLG-HIF-S was purified by linear gradient elution on a Shimadzu LC-2030 using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm). Purity was analyzed using a Shimadzu LC-2030C 3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). Characterization was performed using a Hexin Instruments CMI-1600MALDI-TOF-MS. The purity of the purified peptide was >90.0% at 254 nm.

[0088] III. Synthetic enzyme-sensitive PDC-type PROTAC modified with targeted transmembrane peptides for tumor vasculature and tumor cells

[0089] like Figure 2 As shown, enzyme-sensitive PDC-type PROTAC modified with tumor blood vessel and tumor cell-targeting membrane-penetrating peptides was synthesized using a solid-phase synthesis method based on FMOC amino acids.

[0090] 1. Synthesis of sorafenib hydrolysis product (4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)pyridinecarboxylic acid)

[0091] The synthesis method is the same as in step two.

[0092] 2. Synthesis of Cyclo-A7R-RRR-HIF-S

[0093] 1) Synthesis of Cyclo-A7R-RRR-targeted membrane-penetrating peptide

[0094] Add 50 mg of Fmoc-Arg(Pbf)-Wang resin (0.306 mmol / g, 0.0153 mmol) to a reaction vessel (centrifuge column), add 5 mL of DMF / DCM (1:1), allow to swell for 45 minutes, then filter under reduced pressure and discard the filtrate. Add 20% piperidine / DMF for 10 minutes to remove the FMOC protecting group, filter under reduced pressure, and repeat the above operation twice to completely remove the Fmoc protecting group at the N-terminus of the Fmoc-Arg(Pbf)-Wang resin. After filtration, thoroughly wash the resin with DCM / DMF. Fmoc-Glu-Oall (31.3 mg, 0.0765 mmol), HATU (29.0 mg, 0.0765 mmol), HOBt (11.7 mg, 0.0765 mmol) and DIPEA (36.0 μL, 0.153 mmol) were dissolved in an appropriate amount of DMF. The mixture was added to a centrifuge column and reacted for 45 minutes. The mixture was then filtered and the resin was thoroughly washed with DCM / DMF. Amino acids were sequentially linked according to the sequence shown in SEQ ID NO.8 in Table 1: Fmoc-Pro-OH (25.8 mg, 0.0765 mmol), Fmoc-Leu-OH (27.0 mg, 0.0765 mmol), Fmoc-Arg(Pbf)-OH (51.5 mg, 0.0765 mmol), Fmoc-Arg(Pbf)-OH (51.5 mg, 0.0765 mmol), and Fmoc-Lys(Mtt)-OH (47.8 mg, 0.0765 mmol). Cyclization was then performed using PyBop (39.8 mg, 0.0765 mmol), HOBt (11.7 mg, 0.0765 mmol), and DIPEA (36.0 μL, 0.153 mmol). Two cyclization reactions were performed, one for 3 hours and the other for 8 hours, to obtain the Cyclo-A7R-RRR targeted membrane-penetrating peptide.

[0095] 2) Synthesis of Cyclo-A7R-RRR-HIF-S

[0096] Following the sequence order shown in Table 1 as SEQ ID NO.8, the following amino acids were sequentially linked to the Cyclo-A7R-RRR targeting membrane-penetrating peptide: Fmoc-L-Ile-OH (27.2 mg, 0.0765 mmol), Fmoc-L-Tyr(tBu)-OH (35.3 mg, 0.0765 mmol), Fmoc-4-tert-butoxy-L-proline (31.5 mg, 0.0765 mmol), Fmoc-Ala-OH (22.9 mg, 0.0765 mmol), and Fmoc-Leu-OH (27.2 mg, 0.0765 mmol) to obtain the HIF moiety. Then, mini-PEG (29.6 mg, 0.0765 mmol) was added, and finally, the hydrolysis product of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to a centrifuge column under the action of DIC (17.2 μL, 0.0765 mmol), and the reaction was carried out overnight to link sorafenib to the polypeptide chain. After synthesis, the product was cleaved from the resin using a cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H2O = 90:2.5:2.5:2.5:2.5). DCM was added to the centrifuge column to thoroughly wash the product and the liquid was collected. The solvent was evaporated, ice-cold ether was added to the centrifuge tube, and the product was centrifuged at 8000 rpm for 20 minutes. The supernatant was then discarded, ice-cold ether was added to the centrifuge tube again, the product was gently washed, and the product was centrifuged at 8000 rpm for 5 minutes. Discard the supernatant to obtain the crude peptide Cyclo-A7R-RRR-GFLG-HIF-S, and store it in a -80°C freezer.

[0097] 3) Purification

[0098] The crude peptide was purified by linear gradient elution using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) on a Shimadzu LC-2030. Purity was analyzed using a Shimadzu LC-2030C 3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). Characterization was performed using a Hexin Instruments CMI-1600MALDI-TOF-MS. The purity of the purified peptide was >90.0% at 254 nm.

[0099] 3. Synthesis of Cyclo-A7R-RRR-GFLG-HIF-S

[0100] 1) Synthesis of Cyclo-A7R-RRR-targeted membrane-penetrating peptide

[0101] The synthesis method is the same as step 2.

[0102] 2) Synthesis of Cyclo-A7R-RRR-GFLG

[0103] Following the sequence order shown in Table 1 as SEQ ID NO.9, amino acids were sequentially linked to the Cyclo-A7R-RRR targeting membrane-penetrating peptide: Fmoc-Gly-OH (22.8 mg, 0.0765 mmol), Fmoc-Leu-OH (27.2 mg, 0.0765 mmol), Fmoc-Phe-OH (29.8 mg, 0.0765 mmol), and Fmoc-Gly-OH (22.8 mg, 0.0765 mmol) to obtain Cyclo-A7R-RRR-GFLG.

[0104] 3) Synthesis of Cyclo-A7R-RRR-GFLG-HIF-S

[0105] Following the sequence order shown in Table 1 as SEQ ID NO.9, amino acids were sequentially linked to Cyclo-A7R-RRR-GFLG: Fmoc-L-Ile-OH (27.2 mg, 0.0765 mmol), Fmoc-L-Tyr(tBu)-OH (35.3 mg, 0.0765 mmol), Fmoc-4-tert-butoxy-L-proline (31.5 mg, 0.0765 mmol), Fmoc-Ala-OH (22.9 mg, 0.0765 mmol), and Fmoc-Leu-OH (27.2 mg, 0.0765 mmol) to obtain the HIF moiety. Then, mini-PEG (29.6 mg, 0.0765 mmol) was added, and finally, the hydrolysis product of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to a centrifuge column under the action of DIC (17.2 μL, 0.0765 mmol), and the reaction was carried out overnight to link sorafenib to the polypeptide chain. After synthesis, the product was cleaved from the resin using a cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H2O = 90:2.5:2.5:2.5:2.5). DCM was added to the centrifuge column to thoroughly wash the product and the liquid was collected. The solvent was evaporated, ice-cold ether was added to the centrifuge tube, and the product was centrifuged at 8000 rpm for 20 minutes. The supernatant was then discarded, ice-cold ether was added to the centrifuge tube again, the product was gently washed, and the product was centrifuged at 8000 rpm for 5 minutes. Discard the supernatant to obtain the crude peptide Cyclo-A7R-RRR-GFLG-HIF-S, and store it in a -80°C freezer.

[0106] 4) Purification

[0107] The crude peptide was purified by linear gradient elution using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) on a Shimadzu LC-2030. Purity was analyzed using a Shimadzu LC-2030C 3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). Characterization was performed using a Hexin Instruments CMI-1600MALDI-TOF-MS. The purity of the purified peptide was >90.0% at 254 nm.

[0108] IV. Release Detection

[0109] 1. Detection of PROTAC release from enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides.

[0110] The release of PROTAC from enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides was detected using high-performance liquid chromatography (HPLC). The specific method is as follows:

[0111] The CPP-GFLG-HIF-S synthesized in step two above was used as the experimental group; CPP-GFLG-HIF was used as the negative control group. The sequence of CPP-GFLG-HIF is shown in SEQ ID NO.4 in Table 1. The preparation method of CPP-GFLG-HIF is the same as the synthesis method in step two, except that it is not linked to the sorafenib hydrolysis product.

[0112] Reaction solutions of CPP-GFLG-HIF and CPP-GFLG-HIF-S with a concentration of 1 mg / mL were prepared, and 5 μL of cathepsin B (C-6×His, 1.39 mg / mL, MedChemExpress) was added to each solution. Simultaneously, a 1 mg / mL solution of CPP-GFLG-HIF-S was prepared, with 5 μL of PBS added as a blank control. The three reaction systems were placed at 37°C, and equal amounts of samples were taken at 1, 2, 3, 4, 5, and 6 hours to prepare HPLC samples (Shimadzu LC-2030C 3D Plus). Changes in peak height and peak area of ​​CPP-GFLG-HIF / HIF and CPP-GFLG-HIF-S / HIF-S were observed. Chromatographic conditions: Shimadzu C18 column (C18-120 / 5μm, 10.0×250mm), mobile phase was acetonitrile containing 0.05% TFA and water containing 0.05% TFA, eluted with a gradient of 20%-90% acetonitrile, flow rate: 1ml / min, column temperature: 30℃, detection volume: 50μL, detection wavelength: 254nm.

[0113] The release of PROTAC from enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides, such as... Figures 3 to 5 As shown. From Figure 3 and Figure 4 As can be seen, CPP-GFLG-HIF-S (sequence shown in SEQ ID NO. 3 in Table 1) achieved rapid HIF-S release under the catalysis of cathepsin B, with a release rate of 70.70% within 1 hour, over 90% within 3 hours, and 99.23% within 6 hours. CPP-GFLG-HIF (sequence shown in SEQ ID NO. 4 in Table 1) also achieved rapid HIF release in the presence of cathepsin B, with a release rate of 69.90% within 1 hour, over 90% within 4 hours, and 99.74% within 6 hours. However, without the action of cathepsin B... Figure 5 The release of HIF-S from CPP-GFLG-HIF-S was only 5.28% within 6 hours. These results indicate that the enzyme-sensitive linker (GFLG) can be cleaved under the catalysis of cathepsin B. By linking cell membrane-penetrating peptides to PROTAC through the enzyme-sensitive linker (GFLG), the full release of PROTAC can be achieved.

[0114] 2. Detection of enzyme-sensitive release of PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0115] The release of PROTAC from enzyme-sensitive PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides was detected using high-performance liquid chromatography (HPLC). The specific method is as follows:

[0116] A reaction solution of Cyclo-A7R-RRRR-GFLG-HIF-S with a concentration of 1 mg / mL was prepared, and 5 μL of cathepsin B (C-6×His, 1.39 mg / mL, MedChemExpress) was added to it. The reaction system was placed at 37 °C, and equal amounts of sample were taken at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours to prepare HPLC samples (Shimadzu LC-2030C3D Plus). The changes in peak height and peak area of ​​Cyclo-A7R-RRR-GFLG-HIF-S / HIF-S were observed. Chromatographic conditions: Shimadzu C18 column (C18-120 / 5μm, 10.0×250mm), mobile phase was acetonitrile containing 0.05% TFA and water containing 0.05% TFA, eluted with a gradient of 20%-90% acetonitrile, flow rate: 1ml / min, column temperature: 30℃, detection volume: 50μL, detection wavelength: 254nm.

[0117] The release of PROTAC from enzyme-sensitive PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides, such as... Figure 6 As shown. From Figure 6 As can be seen, Cyclo-A7R-RRR-GFLG-HIF-S (sequence shown as SEQ ID NO.9 in Table 1) achieved rapid release of HIF-S under the catalysis of cathepsin B, with a release rate of 55.41% within 1 hour, nearly 90% of HIF-S released within 4 hours, and 99.15% of HIF-S released within 6 hours. These results indicate that linking tumor vascular and tumor cell-targeting transmembrane peptides to PROTAC via an enzyme-sensitive linker (GFLG) can achieve sufficient release of PROTAC.

[0118] V. Cytotoxicity Detection

[0119] 1. Detection of PDC-type PROTAC cytotoxicity modified with cell-penetrating peptides

[0120] The cytotoxicity of PDC-type PROTAC modified with cell-penetrating peptides against tumor cells and control cells was determined using the MTT assay. The specific method is as follows:

[0121] 1) Cell seeding: HEK293 cells in the growth exponential phase were diluted with DMEM medium to a concentration of 10⁻⁶ cells / mL. 4 Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000–4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0122] 2) Drug administration: 20 μL of different concentrations of HIF-S, CPP-HIF-S, or CPP-GFLG-HIF-S were added to each well, resulting in a final concentration of the compound in the well of 1.0 × 10⁻⁶. -1 μmol / L, 3.0×10 -1 μmol / L, 1.0 μmol / L, 10.0 μmol / L, 50.0 μmol / L, 100.0 μmol / L, experimental groups (CPP-HIF-S, CPP-GFLG-HIF-S) each concentration was set with 3 replicates, negative control (HIF-S) was set with 6 replicates, and continued to be cultured at 37℃ and 5% CO2 for 48h.

[0123] 3) Detection: Add 22 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 10 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate.

[0124] The formula for calculating the cell inhibition rate is:

[0125] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0126] 4) Experimental results: From Figure 7 As can be seen from the data, the cytotoxicity analysis showed that 1.0 × 10 -1 Within a concentration range of μmol / L to 100.0 μmol / L, compounds HIF-S, CPP-HIF-S, and CPP-GFLG-HIF-S had minimal impact on the cell viability of HEK293 cells, with cell survival rates all exceeding 80%, demonstrating that these cell-penetrating peptide-modified PDC-type PROTACs possess a certain degree of safety at the cellular level.

[0127] 2. Detection of PDC-type PROTAC cytotoxicity modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0128] The MTT assay was used to determine the cytotoxicity of PDC-type PROTAC modified with tumor angiogenesis and tumor cell-targeting transmembrane peptides against tumor cells and control cells. The specific method is as follows:

[0129] 1) Cell seeding: HEK293 cells in the growth exponential phase were diluted with DMEM medium to a concentration of 10⁻⁶ cells / mL. 4 Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000–4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0130] 2) Drug administration: 20 μL of different concentrations of HIF-S, Cyclo-A7R-RRRR-HIF-S, or Cyclo-A7R-RRR-GFLG-HIF-S were added to each well, resulting in a final concentration of the compound in the well of 1.0 × 10⁻⁶. -1 μmol / L, 3.0×10 -1μmol / L, 1.0 μmol / L, 10.0 μmol / L, 50.0 μmol / L, 100.0 μmol / L, experimental groups (Cyclo-A7R-RRR-HIF-S, Cyclo-A7R-RRR-GFLG-HIF-S) each concentration were set with 3 replicates, and the negative control (HIF-S) was set with 6 replicates. They were then cultured at 37℃ and 5% CO2 for 48 h.

[0131] 3) Detection: Add 22 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 10 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate.

[0132] The formula for calculating the cell inhibition rate is:

[0133] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0134] 4) Experimental results: From Figure 8 As can be seen from the data, the cytotoxicity analysis showed that 1.0 × 10 -1 Within a concentration range of μmol / L to 100.0 μmol / L, compounds HIF-S, Cyclo-A7R-RRR-HIF-S, or Cyclo-A7R-RRR-GFLG-HIF-S had minimal impact on the cell viability of HEK293 cells, with cell survival rates all exceeding 80%. This demonstrates that these PDC-type PROTACs modified with tumor vasculature and tumor cell-targeting transmembrane peptides possess a certain degree of safety at the cellular level.

[0135] VI. Detection of Tumor Cell Proliferation Inhibition Activity

[0136] 1. Detection of the inhibitory activity of PDC-type PROTAC tumor cells modified with cell-penetrating peptides on cell proliferation.

[0137] U251 and U87 cells in good growth condition and logarithmic growth phase were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in DMEM medium and seeded into 96-well cell culture plates at a seeding density of 5000 cells per well (180 μL). Three wells were prepared for each sample, along with blank wells (no drug administration) and negative controls (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, CPP-HIF-S, and CPP-GFLG-HIF-S were prepared as concentration gradient solutions, and administered to U251 and U87 cells at a dose of 20 μL per well. Forty-eight hours after drug administration, MTT solution was added, and the cells were further incubated at 37°C and 5% CO2 for 4 hours. Cell inhibition rate was calculated after measuring absorbance.

[0138] The formula for calculating the cell inhibition rate is:

[0139] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0140] PDC-type PROTAC tumor cell proliferation inhibitory activity modified with cell-penetrating peptides, such as Figure 6 and Figure 7 As shown.

[0141] from Figure 9 As can be seen, compared with HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S significantly enhanced the inhibitory activity against the proliferation of U251 cells. CPP-GFLG-HIF-S exhibited the best inhibitory activity against U251 cells, increasing the inhibitory activity of HIF-S by 1.68–12.82 times at multiple concentrations. Even at low concentrations (0.1 μmol / L and 0.3 μmol / L), the inhibitory activity of CPP-GFLG-HIF-S against the proliferation of U251 cells was significantly higher than that of HIF-S. These results indicate that the introduction of cell-penetrating peptides can significantly enhance the inhibitory activity of HIF-S against the proliferation of U251 cells. Furthermore, compared with CPP-HIF-S, CPP-GFLG-HIF-S significantly enhanced the inhibitory activity against the proliferation of U251 cells, and the introduction of the enzyme-sensitive linker (GFLG) greatly enhanced the inhibitory activity of PROTAC against the proliferation of U251 cells, and this effect was applicable at all concentrations. Figure 10 The results show that, compared with HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S also significantly enhanced the inhibitory activity on the proliferation of U87 cells, further demonstrating the rationality of the design of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides.

[0142] 2. Detection of the inhibitory activity of PDC-type PROTAC tumor cell proliferation modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0143] U251 and U87 cells in good growth condition and logarithmic growth phase were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in DMEM medium and seeded into 96-well cell culture plates at a seeding density of 5000 cells per well (180 μL). Three wells were prepared for each sample, along with blank wells (no drug administration) and negative controls (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, Cyclo-A7R-RRR-HIF-S, and Cyclo-A7R-RRR-GFLG-HIF-S were prepared as concentration gradient solutions, and administered to U251 and U87 cells at a dose of 20 μL per well. Forty-eight hours after drug administration, MTT solution was added, and the cells were further incubated at 37°C and 5% CO2 for 4 hours. Cell inhibition rate was calculated after measuring absorbance.

[0144] The formula for calculating the cell inhibition rate is:

[0145] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0146] PDC-type PROTAC tumor cell proliferation inhibitory activity modified with tumor vasculature and tumor cell-targeting transmembrane peptides, such as Figure 11 and Figure 12 As shown.

[0147] from Figure 11As can be seen, compared with HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S significantly enhanced the inhibitory activity against the proliferation of U251 cells. Cyclo-A7R-RRR-GFLG-HIF-S exhibited the best inhibitory activity against U251 cell proliferation, increasing the inhibitory activity of HIF-S by 1.78-18.20 times at multiple concentrations. Even at low concentrations (0.1 μmol / L and 0.3 μmol / L), Cyclo-A7R-RRR-HIF-S showed significantly higher inhibitory activity against U251 cell proliferation than HIF-S. These results indicate that the introduction of tumor angiogenesis and tumor cell-targeting transmembrane peptides can significantly enhance the inhibitory activity of HIF-S against the proliferation of U251 cells. Furthermore, compared with Cyclo-A7R-RRR-HIF-S, Cyclo-A7R-RRR-GFLG-HIF-S significantly enhanced the inhibitory activity against the proliferation of U251 cells. The introduction of the enzyme-sensitive linker (GFLG) greatly enhanced the inhibitory activity of PROTAC against the proliferation of U251 cells, and it was applicable at all concentrations. Figure 12 The results show that, compared with HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S also significantly enhanced the inhibitory activity on the proliferation of U87 cells, further demonstrating the rationality of the design of enzyme-sensitive PDC-type PROTAC modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0148] VII. Protein Degradation Activity Assay

[0149] 1. Detection of degradation activity of PDC-type PROTAC protein modified with cell-penetrating peptides

[0150] U251 and U87 cells, which are in good growth condition and in the logarithmic growth phase, were cultured at 10 cells per well. 6 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. Three wells were prepared for each sample, along with blank wells (no drug administration) and negative controls (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, CPP-HIF-S, and CPP-GFLG-HIF-S were selected and prepared as concentration gradient solutions. Forty-eight hours after drug administration, protein samples were collected, and Western blot analysis was performed to investigate the protein degradation activity under different administration methods.

[0151] The degradation activity results of PDC-type PROTAC protein modified with cell-penetrating peptides are as follows: Figures 13 to 14As shown, HIF-S exhibits poor degradation activity against target proteins in U251 cells. At a concentration of 30 μmol / L, it shows no significant degradation effect on BRAF protein in U251 cells, only degrading 28.32% of EphB4 protein, 48.54% of PDGFR-β protein, and 55.91% of VEGFR-2 protein. Similarly, in U87 cells, HIF-S also shows poor degradation activity against target proteins, degrading only 15.51% of VEGFR-2 protein at a concentration of 30 μmol / L. However, when conjugated with cell-penetrating peptides, CPP-HIF-S and CPP-GFLG-HIF-S show significantly enhanced degradation activity against target proteins in U251 cells. In U251 cells, when the concentration of CPP-HIF-S was 1 μmol / L, the degradation rate of VEGFR-2 protein was greater than 50%, while when the concentration was 30 μmol / L, the degradation rates of BRAF protein, EphB4 protein, PDGFR-β protein and VEGFR-2 protein were all greater than 50%. Among them, the degradation rate of VEGFR-2 protein reached 93.15%, and it also had a good degradation effect on target proteins in U87 cells.

[0152] Furthermore, the application of enzyme-sensitive linkers significantly enhances the degradation effect of HIF-S on target proteins. Specifically, in U251 cells, CPP-GFLG-HIF-S exhibits superior degradation effects against BRAF, EphB4, PDGFR-β, and VEGFR-2 proteins compared to CPP-HIF-S. At a concentration of 0.5 μmol / L, CPP-GFLG-HIF-S degraded over 50% of VEGFR-2 protein, achieving a degradation rate 2.08 times higher than CPP-HIF-S at the same concentration. At a concentration of 30 μmol / L, CPP-GFLG-HIF-S showed good degradation effects on both PDGFR-β and VEGFR-2 proteins, with degradation rates of 88.48% and 93.50%, respectively. CPP-GFLG-HIF-S also demonstrated superior degradation effects against target proteins in U87 cells compared to CPP-HIF-S, particularly against VEGFR-2 protein. These results demonstrate that the use of enzyme-sensitive linkers not only does not affect the transmembrane delivery of cell-penetrating peptides to HIF-S, but also enhances the degradation activity of HIF-S on target proteins, further confirming the rationality of the design of enzymatically digestible PDC-PROTACs.

[0153] 2. Detection of degradation activity of PDC-type PROTAC protein modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0154] U251 and U87 cells, which are in good growth condition and in the logarithmic growth phase, were cultured at 10 cells per well.6 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. Three wells were prepared for each sample, along with blank wells (no drug administration) and negative controls (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, Cyclo-A7R-RRR-HIF-S, and Cyclo-A7R-RRR-GFLG-HIF-S were selected and prepared as concentration gradient solutions. Forty-eight hours after drug administration, protein samples were collected, and Western blot analysis was performed to investigate the protein degradation activity under different administration methods.

[0155] Results of the degradation activity of PDC-type PROTAC protein modified with tumor vasculature and tumor cell-targeting transmembrane peptides are as follows: Figure 15 and Figure 16 As shown, HIF-S exhibits poor degradation activity against target proteins in U251 cells. At a concentration of 30 μmol / L, it shows limited effectiveness in degrading BRAF, EphB4, PDGFR-β, and VEGFR-2 proteins in U251 cells. Similarly, in U87 cells, HIF-S also shows poor degradation, degrading only 15.51% of VEGFR-2 protein at a concentration of 30 μmol / L. However, when conjugated with tumor vascular and tumor cell-targeting transmembrane peptides, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S show significantly enhanced degradation activity against target proteins in U251 cells. Cyclo-A7R-RRR-HIF-S exhibited good degradation effects on target proteins in U251 cells, and at high concentrations (10 μmol / L and 30 μmol / L), its degradation effect on PDGFR-β and VEGFR-2 proteins was significantly better than that of HIF-S. Furthermore, Cyclo-A7R-RRR-HIF-S also showed good degradation effects on target proteins in U87 cells.

[0156] Furthermore, the application of enzyme-sensitive linkers significantly enhances the degradation efficiency of HIF-S on target proteins. Specifically, in U251 cells, Cyclo-A7R-RRR-GFLG-HIF-S exhibits superior degradation efficiency against target proteins compared to Cyclo-A7R-RRR-HIF-S. For instance, at a concentration of 5 μmol / L, Cyclo-A7R-RRR-GFLG-HIF-S is 2.38 times more effective than Cyclo-A7R-RRR-HIF-S in degrading EphB4 protein in U251 cells. These results demonstrate that the application of enzyme-sensitive linkers can enhance the degradation activity of HIF-S on target proteins, further confirming the rationale behind the enzymatically hydrolyzable PDC-PROTAC design.

[0157] VIII. Apoptosis Detection

[0158] 1. Apoptosis detection of PDC-type PROTAC cells modified with cell-penetrating peptides

[0159] U251 and U87 cells, which are in good growth condition and in the logarithmic growth phase, were cultured at 10 cells per well. 6 Cells were seeded at a rate of [number] cells per well into 6-well cell culture plates. Three wells were set for each sample, with blank wells (no drug administration) and negative control wells (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, CPP-HIF-S, and CPP-GFLG-HIF-S were selected and prepared as concentration gradient solutions. Forty-eight hours after drug administration, cells were collected and washed with pre-chilled PBS. 100 μL of 1×Combine buffer was added to each tube to resuspend the cells, followed by 5 μL of Annexin V-FITC, and incubation was performed at room temperature in the dark for 10 minutes. 10 μL of propidium iodide solution was added and gently mixed. 400 μL of PBS was added to each tube to resuspend the cells, and apoptosis was detected using flow cytometry (NovoCyte, CytoFLEX).

[0160] Apoptosis detection results of PDC-type PROTAC cells modified with cell-penetrating peptides are as follows: Figures 17 to 19 As shown, the percentage of FITC / PI-positive apoptotic U251 cells treated with CPP-HIF-S and CPP-GFLG-HIF-S increased with increasing concentration. At low concentrations (0.1 μmol / L, 0.5 μmol / L, and 1 μmol / L), the pro-apoptotic effects of CPP-HIF-S and CPP-GFLG-HIF-S on U251 cells showed little difference. However, at high concentrations (10 μmol / L and 30 μmol / L), CPP-GFLG-HIF-S significantly promoted apoptosis in U251 cells, and at a concentration of 30 μmol / L, CPP-GFLG-HIF-S induced apoptosis in 24.95% of U251 cells. This indicates that the introduction of the enzymatic linker enhances the release of HIF-S into U251 cells, promoting U251 cell apoptosis. Furthermore, CPP-GFLG-HIF-S exhibited a stronger pro-apoptotic effect on U87 cells, and at high concentrations, its apoptosis-inducing effect on U87 cells was significantly stronger than that of HIF-S. At a concentration of 30 μmol / L, the percentage of membrane-associated protein FITC / PI-positive apoptotic cells in the CPP-GFLG-HIF-S group was 8.97 times that in the HIF-S group.

[0161] 2. Detection of apoptosis in PDC-type PROTAC cells modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0162] U251 and U87 cells, which are in good growth condition and in the logarithmic growth phase, were cultured at 10 cells per well. 6 Cells were seeded at a rate of [number] cells per well into 6-well cell culture plates. Three wells were set for each sample, with blank wells (no drug administration) and negative control wells (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S, Cyclo-A7R-RRR-HIF-S, and Cyclo-A7R-RRR-GFLG-HIF-S were selected and prepared as concentration gradient solutions. Forty-eight hours after drug administration, cells were collected and washed with pre-chilled PBS. 100 μL of 1×Combine buffer was added to each tube to resuspend the cells, followed by 5 μL of Annexin V-FITC, and incubation was performed at room temperature in the dark for 10 minutes. 10 μL of propidium iodide solution was added and gently mixed. 400 μL of PBS was added to each tube to resuspend the cells, and apoptosis was detected using flow cytometry (NovoCyte, Cyto FLEX).

[0163] The results of apoptosis detection in PDC-type PROTAC cells modified with tumor vasculature and tumor cell-targeting transmembrane peptides are as follows: Figures 20 to 23 As shown, the percentage of FITC / PI-positive apoptotic U251 cells treated with Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S increased with increasing concentration. Cyclo-A7R-RRR-HIF-S had a weaker ability to induce apoptosis in U251 cells; at the same concentration, its pro-apoptotic effect on U251 cells was significantly weaker than that of Cyclo-A7R-RRR-GFLG-HIF-S, indicating that the introduction of the enzymatic linker can increase the release of HIF-S into U251 cells and promote U251 cell apoptosis. Furthermore, Cyclo-A7R-RRR-GFLG-HIF-S had a stronger pro-apoptotic effect on U87 cells. At a concentration of 30 μmol / L, the percentage of FITC / PI-positive apoptotic cells in the Cyclo-A7R-RRR-GFLG-HIF-S group was 11.92 times that in the HIF-S group.

[0164] IX. Cell Cycle Detection

[0165] 1. Cell cycle detection using PDC-type PROTAC modified with cell-penetrating peptides

[0166] U251 cells in optimal growth conditions and logarithmic growth phase were cultured at 10 per well.6 Cells were seeded at a rate of [number] cells per well into 6-well cell culture plates. Three wells were set for each sample, with blank wells (no drug administration) and negative control wells (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-S and CPP-GFLG-HIF-S were selected and prepared as concentration gradient solutions. Forty-eight hours after drug administration, cells were collected by centrifugation at 800g for 5 minutes. The supernatant was carefully removed, and pre-chilled PBS was added to gently resuspend the cells. Cells were collected again by centrifugation. Pre-chilled 70% ethanol was added, and cells were fixed at 4°C for 24 hours. Cells were collected and washed with pre-chilled PBS. 25 μL of propidium iodide staining solution and 10 μL of RNase A solution were added to 0.5 mL of staining buffer (i.e., propidium iodide staining solution). 0.5 mL of propidium iodide staining solution was added to each cell sample, cells were gently resuspended, and incubated at 37°C in the dark for 30 minutes. After staining, the samples were analyzed on a flow cytometer, and the data were analyzed using the NovoExpress software package.

[0167] Cell cycle assay results of PDC-type PROTAC modified with cell-penetrating peptides are as follows: Figures 24 to 25 As shown, compared with HIF-S, the application of CPP-GFLG-HIF-S arrested U251 cells in the S phase, and the proportion of cells arrested in the S phase increased with increasing drug concentration. At a concentration of 10 μmol / L, CPP-GFLG-HIF-S was able to arrest 58.87% of U251 cells in the S phase.

[0168] 2. Cell cycle detection of PDC-type PROTAC cells modified with tumor vasculature and tumor cell-targeting transmembrane peptides.

[0169] U251 cells in optimal growth conditions and logarithmic growth phase were cultured at 10 per well. 6Cells were seeded at a rate of [number] cells per well into 6-well cell culture plates. Three wells were set for each sample, with blank wells (no drug administration) and negative control wells (HIF-S). After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. Cyclo-A7R-RRR-GFLG-HIF-S was selected and prepared as a concentration gradient solution. Forty-eight hours after drug administration, cells were collected by centrifugation at 800g for 5 minutes. The supernatant was carefully removed, and pre-chilled PBS was added to gently resuspend the cells. Cells were collected again by centrifugation. Pre-chilled 70% ethanol was added, and cells were fixed at 4°C for 24 hours. Cells were collected and washed with pre-chilled PBS. 25 μL of propidium iodide staining solution and 10 μL of RNase A solution were added to 0.5 mL of staining buffer (propidium iodide staining solution). 0.5 mL of propidium iodide staining solution was added to each cell sample, cells were gently resuspended, and incubated at 37°C in the dark for 30 minutes. After staining, the samples were analyzed on a flow cytometer, and the data were analyzed using the NovoExpress software package.

[0170] Results of cell cycle assay for PDC-type PROTAC modified with tumor blood vessels and tumor cell-targeting transmembrane peptides: Figure 26 As shown, Cyclo-A7R-RRR-GFLG-HIF-S arrested U251 cells in the S phase, and the proportion of cells arrested in the S phase increased with increasing concentration. At a concentration of 10 μmol / L, Cyclo-A7R-RRR-GFLG-HIF-S was able to arrest 51.52% of U251 cells in the S phase.

[0171] In summary, both enzyme-sensitive PDC-type PROTACs modified with cell-penetrating peptides and enzyme-sensitive PDC-type PROTACs modified with tumor vasculature and tumor cell-targeting membrane-penetrating peptides can be used to prepare anti-tumor drugs (targeting protein degradation and membrane permeation). They have certain targeting and membrane permeation capabilities for human glioma cells, and have little impact on the cell viability of U251 cells, U87 cells, and HEK293 cells. They exhibit proliferative inhibitory activity on U251 cells and U87 cells, can degrade target proteins in U251 cells and U87 cells, can induce apoptosis in U251 cells and U87 cells, and can affect the cell cycle of U251 cells. Modification with cell-penetrating peptides and enzyme-sensitive linkers, as well as modification with tumor vascular and tumor cell-targeting cell-penetrating peptides and enzyme-sensitive linkers, can expand the structural diversity of PROTAC. At the same time, activity assays show that the introduction of cell-penetrating peptides and enzyme-sensitive linkers or tumor vascular and tumor cell-targeting cell-penetrating peptides and enzyme-sensitive linkers plays an important role in improving the protein degradation effect of PROTAC, which can increase the application scenarios of PROTAC and can be regarded as another important area of ​​PROTAC development.

[0172] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, characterized in that, PROTAC was obtained by modifying it with cell-penetrating peptides; The structural formula of the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC is as follows: or 。 2. The method for synthesizing the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 1, characterized in that, Enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides was prepared by artificial solid-phase synthesis based on FMOC amino acids.

3. The application of the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC as described in claim 1 in the preparation of antitumor drug formulations.

4. The application according to claim 3, characterized in that, The anti-tumor drug is an anti-glioma drug.

5. The application according to claim 4, characterized in that, The anti-glioma drug is a drug that targets human brain glioma cells.

6. The application according to claim 3, characterized in that, The concentration of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides was 1.0 × 10⁻⁶. -1 ~100.0 μmol / L.

7. An anti-glioma composition, characterized in that, It includes other active ingredients with anti-glioma activity, the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC of claim 1, and one or more pharmaceutically acceptable carriers or excipients.

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