Cell-penetrating peptide modified enzyme-sensitive PDC type PROTAC as well as preparation method and application of cell-penetrating peptide modified enzyme-sensitive PDC type PROTAC

Abstract

The invention discloses 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 target protein ligand with a cell-penetrating peptide and an enzyme-sensitive Linker (GFLG) as an antitumor drug, has the capability of releasing the PDC type PROTAC under the catalysis of cathepsin B, has small influence on the cell activity of U251 cells, U87 cells and HEK293 cells, has proliferation inhibition activity on the U251 cells and the U87 cells, and can be used for preparing an antitumor drug. The polypeptide can degrade target proteins in U251 cells and U87 cells, can induce apoptosis of the U251 cells and the U87 cells, has an influence on the cycle of the U251 cells, can be used for preparing antitumor drugs (in the aspects of targeted protein degradation and membrane permeation), has a good application prospect in preparation of drugs targeting human brain glioma cells, and can be used in another important field of PROTAC drug development.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeted therapy, and specifically relates to an enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide, and a preparation method and application thereof. Background Art

[0002] Cancer, also known as malignant tumor, is a disease caused by abnormal control of cell division and proliferation. It is a neoplasm formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in abnormal clonal proliferation. This neoplasm often appears as a mass in the local tissue, and cancer cells can also metastasize to other parts of the body along lymphatic vessels or blood vessels to form new tumor metastases. In recent years, the number of new cancer cases has been increasing year by year, which will have a multi-faceted impact on society, including economic burden, consumption 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 (PROTAC) is an emerging drug development technology that degrades specific proteins by utilizing the ubiquitin-proteasome system (UPS) in cells. PROTAC molecules usually consist 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 to each other, PROTAC can mark the target protein as a protein that needs to be degraded, thereby degrading it through the UPS system. When the target protein ligand is an anti-tumor drug, PROTAC can target and degrade a variety of proteins that are closely related to the occurrence and development of tumors, such as nuclear receptor proteins (such as androgen receptor AR and estrogen receptor ER), brominated structures and super-terminal protein families (BET), protein kinases, etc. The degradation of these proteins can inhibit the proliferation and migration of tumor cells and promote their aging and apoptosis. Although PROTAC technology has shown great potential in the field of tumor treatment, due to the large molecular weight and surface area of ​​PROTAC, it has problems of poor solubility and poor membrane permeability in the treatment of tumors. In addition, the design of PROTAC molecules is very challenging and there are potential off-target effects. These problems limit its wide application in clinical practice. Therefore, it is very necessary to further optimize the design of PROTAC molecules.

[0004] Peptide-drug conjugate (PDC) is an emerging targeted therapy that combines the high tissue penetration of peptides with the powerful therapeutic effects of drugs. PDC usually consists of three key parts: targeting peptides (or homing peptides), linkers, and cytotoxic drugs (or payloads). Although PDC drugs have many advantages, their research and development also faces some challenges. For example, peptides have poor stability in the body and are easily degraded by proteases; the synthesis and purification process of PDC drugs is relatively complex and requires high-precision technology and equipment support. Summary of the invention

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

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC is disclosed, which is obtained by modifying PROTAC with a cell-penetrating peptide;

[0008] Wherein, the amino acid sequence of the cell-penetrating peptide is shown as 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 as shown in SEQ ID NO.1.

[0010] More preferably, an enzyme-sensitive linker is also included, and the cell-penetrating peptide and the enzyme-sensitive linker co-modify PROTAC;

[0011] Wherein, the amino acid sequence of the enzyme-sensitive Linker is GFLG.

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

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

[0014] The third aspect of the present invention discloses the use of the above-mentioned cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC in the preparation of anti-tumor drug preparations.

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

[0016] Preferably, the anti-tumor drug is an anti-glioma drug.

[0017] Further preferably, the anti-glioma drug is a drug targeting human brain glioma cells.

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

[0019] The fourth aspect of the present invention discloses an anti-glioma composition, characterized in that it comprises other active ingredients with anti-glioma effects, 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] The present invention provides a cell-penetrating peptide modified enzyme-sensitive PDC-type PROTAC, 1) combining PDC and PROTAC design strategies, using cell-penetrating peptides to modify PROTACs with anti-tumor ability, which can significantly improve the anti-tumor cell proliferation activity, protein degradation effect and apoptosis-promoting ability of PROTAC, and has high efficiency, and can reduce the amount of PDC-type PROTAC. 2) When the cell-penetrating peptide with the amino acid sequence shown in SEQ ID NO.10 is used, the modified PROTAC has better release than the unmodified PROTAC, indicating that it has better permeability; 3) When the cell-penetrating peptide with the amino acid sequence shown in SEQ ID NO.11 is used, the modified PROTAC has better tumor vascular and tumor cell targeting and membrane penetration effects than the unmodified PROTAC; 4) The modified PROTAC has certain safety at the cellular level; 5) The modified PROTAC has significantly improved tumor proliferation inhibition activity and better target protein degradation effect than the unmodified PROTAC, proving the rationality of the design strategy of this method, and can solve the off-target effect of traditional PROTAC. 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 brain glioma cells, and can serve as another important area of ​​PROTAC drug development.

[0022] Furthermore, PROTAC modified with cell-penetrating peptide and enzyme-sensitive linker has better effect than PROTAC modified with cell-penetrating peptide alone, specifically: 1) enzyme-sensitive linker has the ability to release PROTAC under the catalysis of cathepsin B, which can achieve cell penetration and protein degradation effects at the same time; 2) it has little effect on the cell viability of U251 cells, U87 cells and HEK293 cells; 3) it has proliferation inhibitory activity on U251 cells and U87 cells, and the proliferation inhibitory activity is significantly better than PDC-type PROTAC; 4) it can degrade the target protein in U251 cells and U87 cells, and can induce apoptosis of U251 cells and U87 cells, and affect the cell cycle of U251. Therefore, the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide can be used to prepare anti-tumor drugs (especially targeted protein degradation and membrane penetration), and has good application prospects in the preparation of drugs targeting human glioma cells, which can be used as another important field of PROTAC drug development.

[0023] The preparation method of the cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC provided by the present invention is prepared by chemical synthesis, has a mature technical route, is easy to prepare in large quantities, has good stability, and has potential application prospects for the current rare PDC-type PROTAC research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of the synthesis method of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides;

[0025] Figure 2 Flow chart of the synthesis method of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides targeting tumor blood vessels and tumor cells;

[0026] Figure 3 This is a diagram of the PROTAC release process in the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide (in the presence of enzyme);

[0027] Figure 4 This is a diagram of the Linker cleavage process in the enzyme-sensitive PDC-type PROTAC control modified with a cell-penetrating peptide (in the presence of enzyme);

[0028] Figure 5 This is a diagram of the PROTAC release process in the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide (in the absence of enzyme);

[0029] Figure 6 A diagram of the PROTAC release process in an enzyme-sensitive PDC-type PROTAC modified with a transmembrane peptide targeting tumor blood vessels and tumor cells (in the presence of an enzyme);

[0030] Figure 7 This is a result diagram showing the effect of PDC-type PROTAC modified with cell-penetrating peptide on the viability of HEK293 cells;

[0031] Figure 8 This is a graph showing the effect of PDC-type PROTAC modified with transmembrane peptides targeting tumor blood vessels and tumor cells on the viability of HEK293 cells;

[0032] Fig. 9 This is a graph showing the inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptide on U251 cell proliferation;

[0033] Fig.10 This is a graph showing the inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptide on U87 cell proliferation;

[0034] Fig.11 This is a graph showing the inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptides targeting tumor blood vessels and tumor cells on U251 cell proliferation;

[0035] Fig.12 This is a graph showing the inhibitory activity of PDC-type PROTAC modified with transmembrane peptides targeting tumor blood vessels and tumor cells on the proliferation of U87 cells;

[0036] Fig.13 The results of the degradation effect of PDC-type PROTAC modified with cell-penetrating peptide on the target protein in U251 cells are shown in Figure 1; wherein: (A) is the result of the degradation effect of HIF-S on the target protein in U251 cells, (C) is the result of the degradation effect of CPP-HIF-S on the target protein in U251 cells, (E) is the result of the degradation effect of CPP-GFLG-HIF-S on the target protein in U251 cells, (B), (D) and (F) are the semi-quantitative results of the target protein expression levels in (A), (C) and (E), respectively;

[0037] Fig.14 The results of the degradation effect of PDC-type PROTAC modified with cell-penetrating peptide on the target protein in U87 cells are shown in Figure 1; wherein: (A) is the result of the degradation effect of HIF-S on the target protein in U87 cells, (C) is the result of the degradation effect of CPP-HIF-S on the target protein in U87 cells, (E) is the result of the degradation effect of CPP-GFLG-HIF-S on the target protein in U87 cells, (B), (D) and (F) are the semi-quantitative results of the target protein expression levels in (A), (C) and (E), respectively;

[0038] Fig.15The results of the degradation effect of PDC-type PROTAC modified with tumor vascular and tumor cell-targeted transmembrane peptides on the target protein in U251 cells are shown in Figure 1; wherein: (A) is the result of the degradation effect of Cyclo-A7R-RRR-HIF-S on the target protein in U251 cells, (C) is the result of the degradation effect of Cyclo-A7R-RRR-GFLG-HIF-S on the target protein in U251 cells, (B) and (D) are the semi-quantitative results of the target protein expression levels in (A) and (C);

[0039] Fig.16 The results of the degradation effect of PDC-type PROTAC modified with tumor vascular and tumor cell-targeted transmembrane peptides on the target protein in U87 cells are shown in Figure 1; wherein: (A) is the result of the degradation effect of Cyclo-A7R-RRR-HIF-S on the target protein in U87 cells, (C) is the result of the degradation effect of Cyclo-A7R-RRR-GFLG-HIF-S on the target protein in U87 cells, (B) and (D) are the semi-quantitative results of the target protein expression levels in (A) and (C);

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

[0041] Fig.18 The effect diagram of the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide on the apoptosis of U251 cells; wherein: (A) is the effect of CPP-GFLG-HIF-S on the apoptosis of U251 cells, and (B) is the quantitative result of apoptotic cells in (A);

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

[0043] Fig. 20 The effect diagram of the PDC-type PROTAC modified with tumor vascular and tumor cell-targeted transmembrane peptides on the apoptosis of U251 cells; (A) is the effect of Cyclo-A7R-RRR-HIF-S on the apoptosis of U251 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0044] Fig.21The effect diagram of the enzyme-sensitive PDC-type PROTAC modified with tumor vascular and tumor cell-targeted transmembrane peptides on the apoptosis of U251 cells; wherein: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on the apoptosis of U251 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0045] Fig. 22 The effect diagram of PDC-type PROTAC on U87 cell apoptosis; (A) is the effect of HIF-S on U251 cell apoptosis, (B) is the quantitative result of apoptotic cells in (A);

[0046] Fig.23 The effect diagram of the enzyme-sensitive PDC-type PROTAC modified with tumor vascular and tumor cell-targeted transmembrane peptides on the apoptosis of U87 cells; wherein: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on the apoptosis of U87 cells, and (B) is the quantitative result of apoptotic cells in (A);

[0047] Fig.24 The effect diagram of PDC-type PROTAC on the U251 cell cycle; wherein: (A) is the effect of HIF-S on the U251 cell cycle, (B) is the quantitative result of (A);

[0048] Fig.25 The effect diagram of the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide on the U251 cell cycle; wherein: (A) is the effect of CPP-GFLG-HIF-S on the U251 cell cycle, and (B) is the quantitative result of (A);

[0049] Fig.26 The effect diagram of the enzyme-sensitive PDC-type PROTAC modified with transmembrane peptide targeting tumor blood vessels and tumor cells on the U251 cell cycle; wherein: (A) is the effect of Cyclo-A7R-RRR-GFLG-HIF-S on the U251 cell cycle, and (B) is the quantitative result of (A). DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

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

[0052] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0053] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0054] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

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

[0056] The present invention provides a method for synthesizing a cell-penetrating peptide-modified PDC-type PROTAC, wherein a hypoxia-inducible factor 1α (HIF-1α) protein fragment (the sequence is shown in SEQ ID NO.6 in Table 1) and sorafenib are connected by mini-PEG to obtain a PROTAC (the sequence is shown in SEQ ID NO.1 in Table 1); and then the PROTAC is modified with a cell-penetrating peptide to obtain a cell-penetrating peptide-modified PDC-type PROTAC;

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

[0058] Furthermore, a cell-penetrating peptide and an enzyme-sensitive linker (GFLG) can be used to co-modify PDC-type PROTAC to obtain a cell-penetrating peptide-modified PDC-type PROTAC.

[0059] Table 1 Amino Acid Sequence Listing

[0060]

[0061]

[0062] Note: "L" in the amino acid sequence stands for "Leucine", "A" stands for "Alanine", "P(OH)" stands for "Hydroxy-L-Proline", "Y" stands for "Tyrosine", "I" stands for "Isoleucine", "S" stands for "Sorafenib", "C(……)" stands for cyclization, "f" stands for "D-Phenylalanine", "Φ" stands for "L-2-Naphthylalanine", "R" stands for "L-Arginine", "r" stands for "D-Arginine", "E" stands for "Glutamic Acid", "G" stands for "Glycine", "F" stands for "L-Phenylalanine", "K" stands for "Lysine", and "P" stands for "Proline".

[0063] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0064] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, "%" means weight percentage unless otherwise specified.

[0065] 1. Experimental Materials

[0066] 1. Experimental drugs and reagents: Rink Amide-MBHA resin (B&K), Fmoc-Arg(Pbf)-Wang resin (B&K), Fmoc-protected amino acid, piperidine (Sinopharm), DMF (Kermione Chemical Reagent), DCM (Kermione Chemical Reagent), HATU (Biode Pharmaceuticals), HOBt (Biode Pharmaceuticals), DIPEA (Biode Pharmaceuticals), PyBop (Biode Pharmaceuticals), TFA (Aladdin), TIS (Aladdin), 1,3-dimethoxybenzene (Aladdin), DODT (Aladdin), chromatography 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. Experimental cells: U251 (human glioma cells), U87 (human glioma cells) and HEK293 (human embryonic kidney cells).

[0068] 2. Synthetic cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC

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

[0070] 1. Synthesis of Sorafenib Hydrolysis Product (4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinic acid)

[0071] Sorafenib (1.00 g, 2.15 mmol) and NaOH (1.40 g, 32.27 mmol) were dissolved in anhydrous ethanol and heated at 80 °C with N 2 The reaction was protected for 10 hours. Excess ethanol was separated and dilute hydrochloric acid (2 mol / L) was added to neutralize the excess alkali to make the pH value = 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 transmembrane peptide

[0074] Take Rink Amide-MBHA resin (50.0 mg, 0.32 mmol / g, 0.016 mmol) into a reaction vessel (centrifugal column), add 5 mL DMF / DCM (1:1), swell for 45 minutes, and then discard the filtrate by vacuum filtration. Add 20% piperidine / DMF for 10 minutes to remove the FMOC protecting group, and filter the filtrate under reduced pressure. Repeat the above operation twice to completely remove the Fmoc protecting group at the N-terminus of the Rink Amide-MBHA resin. After filtration, wash the Rink Amide-MBHA resin thoroughly 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 mixed liquid was added to the centrifugal column, reacted for 45 minutes, filtered and the resin was thoroughly washed with DCM / DMF. The amino acids were connected in sequence according to the sequence order shown in SEQ ID NO. 2 in Table 1: 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-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). PyBop (41.6 mg, 0.08 mmol), HOBt (12.2 mg, 0.08 mmol) and DIPEA (37.6 μL, 0.16 mmol) were used for cyclization. The cyclization reaction was carried out twice, for 3 hours and 8 hours respectively, to obtain the CPP transmembrane peptide.

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

[0076] According to the sequence order shown in SEQ ID NO. 2 in Table 1, the following amino acids were connected 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 part. Then mini-PEG (30.8 mg, 0.08 mmol) was connected, and finally, under the action of N, N-diisopropylcarbodiimide (DIC; 17.9 μL, 0.08 mmol), the hydrolyzate of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to the centrifugal column and reacted overnight to connect sorafenib to the polypeptide chain. After the synthesis, the cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H 2 The product was cleaved from the resin using 1% paraformaldehyde (0=90:2.5:2.5:2.5:2.5:2.5). DCM was added to the centrifuge column to thoroughly wash the product and collect the liquid. The solvent was evaporated, icy ether was added to the centrifuge tube, the product was centrifuged at 8000 rpm for 20 minutes, and then the supernatant was discarded. The icy ether was added to the centrifuge tube again, the product was gently washed, and centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded to obtain the crude peptide CPP-HIF-S, which was stored in a -80°C refrigerator.

[0077] 3) Purification

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

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

[0080] 1) Synthesis of CPP transmembrane peptide

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

[0082] 2) Synthesis of CPP-GFLG

[0083] According to the sequence order shown in SEQ ID NO.3 in Table 1, the following amino acids were connected 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] According to the sequence order shown in SEQ ID NO.3 in Table 1, amino acids 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) were sequentially connected to CPP-GFLG to obtain the HIF part. Then mini-PEG (30.8 mg, 0.08 mmol) was connected. Finally, under the action of DIC (17.9 μL, 0.08 mmol), the hydrolyzate of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to the centrifugal column and reacted overnight to connect sorafenib to the polypeptide chain. After the synthesis, the cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H 2 The product was cleaved from the resin using 1% paraformaldehyde (0=90:2.5:2.5:2.5:2.5:2.5). DCM was added to the centrifuge column to thoroughly wash the product and collect the liquid. The solvent was evaporated, icy ether was added to the centrifuge tube, the product was centrifuged at 8000 rpm for 20 minutes, and then the supernatant was discarded. The icy ether was added to the centrifuge tube again, 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 in a -80°C refrigerator.

[0086] 4) Purification

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

[0088] 3. Synthesis of enzyme-sensitive PDC-type PROTACs modified with cell-penetrating peptides targeting tumor vasculature and tumor cells

[0089] like Figure 2 As shown, an enzyme-sensitive PDC-type PROTAC modified with a tumor vascular and tumor cell-targeting transmembrane peptide 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)picolinic acid)

[0091] The synthesis method is the same as step 2.

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

[0093] 1) Synthesis of Cyclo-A7R-RRR targeting transmembrane peptide

[0094] Take Fmoc-Arg(Pbf)-Wang resin (50 mg, 0.306 mmol / g, 0.0153 mmol) into a reaction vessel (centrifugal column), add 5 mL DMF / DCM (1:1), swell for 45 minutes, and then discard the filtrate by vacuum filtration. Add 20% piperidine / DMF for 10 minutes to remove the FMOC protecting group, and filter the filtrate under reduced pressure. 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, wash the resin thoroughly 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 mixed liquid was added to the centrifugal column, reacted for 45 minutes, filtered and the resin was thoroughly washed with DCM / DMF. The amino acids 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), Fmoc-Arg(Pbf)-OH (51.5 mg, 0.0765 mmol), and Fmoc-Lys(Mtt)-OH (47.8 mg, 0.0765 mmol) were connected in sequence according to the sequence order of SEQ ID NO.8 in Table 1. PyBop (39.8 mg, 0.0765 mmol), HOBt (11.7 mg, 0.0765 mmol) and DIPEA (36.0 μL, 0.153 mmol) were then used for cyclization. The cyclization reaction was carried out twice, for 3 hours and 8 hours respectively, to obtain the Cyclo-A7R-RRR targeting transmembrane peptide.

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

[0096] According to the sequence order shown in SEQ ID NO. 8 in Table 1, the amino acids 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) were sequentially connected to the Cyclo-A7R-RRR targeting transmembrane peptide to obtain the HIF portion. Then mini-PEG (29.6 mg, 0.0765 mmol) was connected, and finally, under the action of DIC (17.2 μL, 0.0765 mmol), the hydrolyzate of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to the centrifugal column and reacted overnight to connect sorafenib to the polypeptide chain. After the synthesis was completed, the cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H 2 The product was cleaved from the resin by filtration at 40 °C (20 °C) and 50 °C (40 °C) for 24 hours. DCM was added to the centrifuge column to thoroughly wash the product and collect the liquid. The solvent was evaporated, icy ether was added to the centrifuge tube, the product was centrifuged at 8000 rpm for 20 minutes, and then the supernatant was discarded. The icy ether was added to the centrifuge tube again, the product was gently washed, and centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded to obtain the crude peptide Cyclo-A7R-RRR-GFLG-HIF-S, which was stored in a -80 °C refrigerator.

[0097] 3) Purification

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

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

[0100] 1) Synthesis of Cyclo-A7R-RRR targeting transmembrane peptide

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

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

[0103] According to the sequence order shown in SEQ ID NO.9 in Table 1, the amino acids 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) were sequentially connected to the Cyclo-A7R-RRR targeting transmembrane peptide to obtain Cyclo-A7R-RRR-GFLG.

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

[0105] According to the sequence order shown in SEQ ID NO. 9 in Table 1, the following amino acids were connected to Cyclo-A7R-RRR-GFLG in sequence: 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 part. Then mini-PEG (29.6 mg, 0.0765 mmol) was connected, and finally, under the action of DIC (17.2 μL, 0.0765 mmol), the hydrolyzate of sorafenib obtained in step 1 (45.2 mg, 0.10 mmol) was added to the centrifugal column and reacted overnight to connect sorafenib to the polypeptide chain. After the synthesis was completed, the cleavage reagent (TFA:TIS:1,3-dimethoxybenzene:DODT:H 2 The product was cleaved from the resin by filtration at 40 °C (20 °C) and 50 °C (40 °C) for 24 hours. DCM was added to the centrifuge column to thoroughly wash the product and collect the liquid. The solvent was evaporated, icy ether was added to the centrifuge tube, the product was centrifuged at 8000 rpm for 20 minutes, and then the supernatant was discarded. The icy ether was added to the centrifuge tube again, the product was gently washed, and centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded to obtain the crude peptide Cyclo-A7R-RRR-GFLG-HIF-S, which was stored in a -80 °C refrigerator.

[0106] 4) Purification

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

[0108] 4. Release Test

[0109] 1. Release detection of PROTAC in enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides

[0110] The release of PROTAC in the prepared cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC was detected by high performance liquid chromatography. The specific method is as follows:

[0111] The CPP-GFLG-HIF-S synthesized in the above step 2 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 refers to the synthesis method in step 2, except that the sorafenib hydrolysate is not connected.

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

[0113] The release of PROTAC in the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides is as follows Figures 3 to 5 As shown. Figure 3 and Figure 4 It can be seen that CPP-GFLG-HIF-S (sequence as shown in SEQ ID NO.3 in Table 1) achieves rapid release of HIF-S under the catalysis of cathepsin B, with the release amount reaching 70.70% within 1 hour, more than 90% of HIF-S can be released within 3 hours, and 99.23% of HIF-S can be released within 6 hours. CPP-GFLG-HIF (sequence as shown in SEQ ID NO.4 in Table 1) also achieves rapid release of HIF in the presence of cathepsin B, with the release amount reaching 69.90% within 1 hour, more than 90% of HIF can be released within 4 hours, and 99.74% of HIF can be released within 6 hours. In the absence of cathepsin B ( Figure 5 ), the release of HIF-S in 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, and the cell-penetrating peptide can be connected to PROTAC through the enzyme-sensitive linker (GFLG), which can achieve the full release of PROTAC.

[0114] 2. Release detection of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide targeting tumor blood vessels and tumor cells

[0115] High performance liquid chromatography was used to detect the release of PROTAC in the prepared tumor vascular and tumor cell targeting transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC. The specific method is as follows:

[0116] A reaction solution of Cyclo-A7R-RRR-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 thereto. The reaction system was placed at 37°C, and equal amounts of samples 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 the peak height and peak area of ​​Cyclo-A7R-RRR-GFLG-HIF-S / HIF-S were observed respectively. Chromatographic conditions: Shimadzu C18 column (C18-120 / 5 μm, 10.0×250 mm), mobile phase consisting of acetonitrile containing 0.05% TFA and water containing 0.05% TFA, gradient elution with 20%-90% acetonitrile, flow rate: 1 ml / min, column temperature: 30°C, detection volume: 50 μL, detection wavelength: 254 nm.

[0117] The release of PROTAC in the enzyme-sensitive PDC-type PROTAC modified with transmembrane peptides targeting tumor blood vessels and tumor cells is as follows Figure 6 As shown. Figure 6 It can be seen that Cyclo-A7R-RRR-GFLG-HIF-S (sequence as shown in SEQ ID NO.9 in Table 1) achieves rapid release of HIF-S under the catalysis of cathepsin B, with a release of 55.41% within 1 hour, and nearly 90% of HIF-S can be released in 4 hours, and 99.15% of HIF-S can be released within 6 hours. These results show that connecting tumor vascular and tumor cell targeting transmembrane peptides to PROTAC through enzyme-sensitive Linker (GFLG) can achieve full release of PROTAC.

[0118] 5. Cytotoxicity Assay

[0119] 1. Cytotoxicity assay of PDC-type PROTAC modified with cell-penetrating peptides

[0120] The MTT method was used to determine the cytotoxicity of PDC-type PROTAC modified with cell-penetrating peptides on tumor cells and control cells. The specific method is as follows:

[0121] 1) Cell inoculation: HEK293 cells in the exponential growth phase were diluted to 10 4 Cell solutions of the order of 100 cells / mL were inoculated in parallel in 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and incubated at 37°C, 5% CO 2 Culture for 12 h.

[0122] 2) Drug administration: 20 μL of HIF-S, CPP-HIF-S or CPP-GFLG-HIF-S of different concentrations were added to each well to obtain a final concentration of the compound in the well of 1.0×10 -1 μmol / L, 3.0×10 -1 μmol / L, 1.0μmol / L, 10.0μmol / L, 50.0μmol / L, 100.0μmol / L, three replicate wells were set for each concentration of the experimental group (CPP-HIF-S, CPP-GFLG-HIF-S), and six replicate wells were set for the negative control (HIF-S). The cells were kept at 37°C and 5% CO 2 Culture 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 MTT in each well. Incubate at 37°C and 5% CO 2After incubation for 4 h, the supernatant was carefully removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min. The UV absorbance (OD value) of each well at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) and the cell inhibition rate was calculated.

[0124] The calculation formula of cell inhibition rate is:

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

[0126] 4) Experimental results: Figure 7 As can be seen, the cytotoxicity analysis showed that 1.0×10 -1 In the concentration range of 1 μmol / L to 100.0 μmol / L, the compounds HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S had little effect on the cell viability of HEK293 cells, and the cell survival rates were all above 80%, proving that this type of cell-penetrating peptide-modified PDC-type PROTAC has a certain safety at the cellular level.

[0127] 2. Detection of cytotoxicity of PDC-type PROTACs modified with cell-penetrating peptides targeting tumor blood vessels and tumor cells

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

[0129] 1) Cell inoculation: HEK293 cells in the exponential growth phase were diluted to 10 4 Cell solutions of the order of 100 cells / mL were inoculated in parallel in 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and incubated at 37°C, 5% CO 2 Culture for 12 h.

[0130] 2) Administration: 20 μL of HIF-S, Cyclo-A7R-RRR-HIF-S or Cyclo-A7R-RRR-GFLG-HIF-S of different concentrations was added to each well to obtain a final concentration of the compound in the well of 1.0×10 -1 μmol / L, 3.0×10 -1 μmol / L, 1.0μmol / L, 10.0μmol / L, 50.0μmol / L, 100.0μmol / L, three replicate wells for each concentration of the experimental group (Cyclo-A7R-RRR-HIF-S, Cyclo-A7R-RRR-GFLG-HIF-S), and six replicate wells for the negative control (HIF-S). Continue to incubate at 37°C, 5% CO 2Culture for 48h.

[0131] 3) Detection: Add 22 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL MTT in each well. Incubate at 37°C and 5% CO 2 After incubation for 4 h, the supernatant was carefully removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min. The UV absorbance (OD value) of each well at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) and the cell inhibition rate was calculated.

[0132] The calculation formula of cell inhibition rate is:

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

[0134] 4) Experimental results: Figure 8 As can be seen, the cytotoxicity analysis showed that 1.0×10 -1 In the concentration range of μmol / L to 100.0 μmol / L, the compounds HIF-S, Cyclo-A7R-RRR-HIF-S or Cyclo-A7R-RRR-GFLG-HIF-S had little effect on the cell viability of HEK293 cells, and the cell survival rate was above 80%, proving that this type of tumor vascular and tumor cell-targeting transmembrane peptide-modified PDC-type PROTAC has a certain safety at the cellular level.

[0135] 6. Tumor Cell Proliferation Inhibitory Activity Detection

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

[0137] U251 cells and U87 cells in good growth state and logarithmic growth phase were centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in DMEM medium and inoculated into a 96-well cell culture plate at an inoculation volume of 5000 cells per well, with an inoculation volume of 180μL. Three wells were set for each sample, and blank wells (no drug administration) and negative controls (HIF-S) were set. After inoculation, the culture plate containing the cells was placed at 37°C and 5% CO 2 HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution, and administered to U251 cells and U87 cells at a dose of 20 μL per well. After 48 hours of administration, MTT solution was added and then placed at 37°C, 5% CO 2 The cells were further cultured in an incubator for 4 hours. The cell inhibition rate was calculated after measuring the absorbance.

[0138] The calculation formula of cell inhibition rate is:

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

[0140] The inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptide on tumor cell proliferation Figure 6 and Figure 7 shown.

[0141] from Fig. 9 It can be seen that compared with HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S have significantly improved the proliferation inhibitory activity of U251 cells. Among them, CPP-GFLG-HIF-S has the best proliferation inhibitory activity on U251 cells, and at multiple concentrations, it can increase the proliferation inhibitory activity of HIF-S by 1.68 to 12.82 times. At low concentrations (concentrations of 0.1 μmol / L and 0.3 μmol / L), the proliferation inhibitory activity of CPP-GFLG-HIF-S on U251 cells is also significantly higher than that of HIF-S. These results show that the introduction of cell-penetrating peptides can significantly enhance the proliferation inhibitory activity of HIF-S on U251 cells. In addition, compared with CPP-HIF-S, the proliferation inhibitory activity of CPP-GFLG-HIF-S on U251 cells is significantly enhanced, and the introduction of enzyme-sensitive Linker (GFLG) greatly enhances the proliferation inhibitory activity of PROTAC on U251 cells, and is applicable at all concentrations. Fig.10 The results shown in the figure indicate that, compared with HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S also had significantly enhanced proliferation inhibitory activity against U87 cells, further demonstrating the rationality of the design of enzyme-sensitive PDC-type PROTACs modified with cell-penetrating peptides.

[0142] 2. Detection of tumor cell proliferation inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptides targeting tumor blood vessels and tumor cells

[0143] U251 cells and U87 cells in good growth state and logarithmic growth phase were centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in DMEM medium and inoculated into a 96-well cell culture plate at an inoculation volume of 5000 cells per well, with an inoculation volume of 180μL. Three wells were set for each sample, and blank wells (no drug administration) and negative controls (HIF-S) were set. After inoculation, the culture plate containing the cells was placed at 37°C and 5% CO 2HIF-S, Cyclo-A7R-RRR-HIF-S, and Cyclo-A7R-RRR-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution, and administered to U251 cells and U87 cells at a dose of 20 μL per well. After 48 hours of administration, MTT solution was added and then placed at 37°C, 5% CO 2 The cells were further cultured in an incubator for 4 hours. The cell inhibition rate was calculated after measuring the absorbance.

[0144] The calculation formula of cell inhibition rate is:

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

[0146] Tumor cell proliferation inhibition activity of PDC-type PROTAC modified with tumor vascular and tumor cell-targeting transmembrane peptides Fig.11 and Fig.12 shown.

[0147] from Fig.11 It can be seen that compared with HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S significantly improved the proliferation inhibitory activity of U251 cells. Among them, Cyclo-A7R-RRR-GFLG-HIF-S has the best proliferation inhibitory activity on U251 cells, and at multiple concentrations, it can increase the proliferation inhibitory activity of HIF-S by 1.78-18.20 times. At low concentrations (concentrations of 0.1μmol / L and 0.3μmol / L), the proliferation inhibitory activity of Cyclo-A7R-RRR-HIF-S on U251 cells is also significantly higher than that of HIF-S. These results show that the introduction of tumor vascular and tumor cell-targeted transmembrane peptides can significantly enhance the proliferation inhibitory activity of HIF-S on U251 cells. In addition, compared with Cyclo-A7R-RRR-HIF-S, Cyclo-A7R-RRR-GFLG-HIF-S significantly improved the proliferation inhibitory activity of U251 cells. The introduction of the enzyme-sensitive linker (GFLG) greatly improved the proliferation inhibitory activity of PROTAC on U251 cells, and was applicable at all concentrations. Fig.12 The results shown in the figure indicate that compared with HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S also significantly increased the proliferation inhibitory activity of U87 cells, further demonstrating the rationality of the design of enzyme-sensitive PDC-type PROTACs modified with transmembrane peptides targeting tumor blood vessels and tumor cells.

[0148] VII. Protein degradation activity detection

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

[0150] U251 cells and U87 cells in good growth state and logarithmic growth phase were cultured at 10 per well. 6 The cells were inoculated into a 6-well cell culture plate at a density of 100 cells / well. Three wells were set for each sample, and blank wells (no drug administration) and negative control (HIF-S) were set. After inoculation, the culture plate containing cells was placed at 37°C and 5% CO 2 The cells were incubated in a constant temperature incubator for 24 hours. HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution. After 48 hours of administration, protein samples were collected and the protein degradation activity of different administration methods was studied by western blot.

[0151] The results of protein degradation activity of PDC-type PROTAC modified with cell-penetrating peptides are as follows Figure 13 to Figure 14 As shown, it can be seen that HIF-S has poor degradation activity on the target protein in U251 cells. When its concentration is 30 μmol / L, it has no obvious degradation effect on BRAF protein in U251 cells, and can only degrade 28.32% of EphB4 protein, 48.54% of PDGFR-β protein and 55.91% of VEGFR-2 protein. In U87 cells, HIF-S also has poor degradation effect on the target protein. When its concentration is 30 μmol / L, it can only degrade 15.51% of VEGFR-2 protein. After connecting with cell-penetrating peptides, the degradation activity of CPP-HIF-S and CPP-GFLG-HIF-S on the target protein in U251 cells is significantly improved. 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%, and when its 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 which the degradation rate of VEGFR-2 protein reached 93.15%, and it also had a good degradation effect on the target protein in U87 cells.

[0152] Furthermore, the use of enzyme-sensitive Linker can significantly improve the degradation effect of HIF-S on target proteins. Specifically, in U251 cells, CPP-GFLG-HIF-S has better degradation effects on BRAF protein, EphB4 protein, PDGFR-β protein and VEGFR-2 protein than CPP-HIF-S. When the concentration of CPP-GFLG-HIF-S is 0.5μmol / L, more than 50% of VEGFR-2 protein can be degraded, and the degradation rate of VEGFR-2 protein at the same concentration is 2.08 times that of CPP-HIF-S. When the concentration of CPP-GFLG-HIF-S is 30μmol / L, the degradation effects on PDGFR-β protein and VEGFR-2 protein are good, with degradation rates of 88.48% and 93.50%, respectively. CPP-GFLG-HIF-S is also better than CPP-HIF-S in the degradation of target proteins in U87 cells, especially in the degradation of VEGFR-2 protein. These results indicate that the use of enzyme-sensitive linkers will not only not affect the transmembrane delivery of cell-penetrating peptides to HIF-S, but also enhance the degradation activity of HIF-S on target proteins, further confirming the rationality of the design of enzymatic PDC-PROTACs.

[0153] 2. Detection of protein degradation activity of PDC-type PROTAC modified with cell-penetrating peptides targeting tumor blood vessels and tumor cells

[0154] U251 cells and U87 cells in good growth state and logarithmic growth phase were cultured at 10 per well. 6 The cells were inoculated into 6-well cell culture plates at a density of 100 cells / well. Three wells were set for each sample, and blank wells (no drug) and negative control (HIF-S) were set. After inoculation, the culture plates containing cells were placed at 37°C and 5% CO 2 The cells were incubated in a constant temperature incubator for 24 hours. HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution. After 48 hours of administration, protein samples were collected and the protein degradation activity of different administration methods was studied by western blot.

[0155] The results of protein degradation activity of PDC-type PROTAC modified with cell-penetrating peptide targeting tumor blood vessels and tumor cells are as follows Fig.15 and Fig.16As shown, it can be seen that HIF-S has poor degradation activity on target proteins in U251 cells. When its concentration is 30 μmol / L, the degradation effect on BRAF protein, EphB4 protein, PDGFR-β protein and VEGFR-2 protein in U251 cells is poor. In U87 cells, HIF-S also has poor degradation effect on target proteins. When its concentration is 30 μmol / L, it can only degrade 15.51% of VEGFR-2 protein. After being connected with tumor vascular and tumor cell targeting transmembrane peptides, the degradation activity of Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S on target proteins in U251 cells was significantly improved. Cyclo-A7R-RRR-HIF-S has a good degradation effect on the target protein in U251 cells, and at high concentrations (10μmol / L and 30μmol / L), the degradation effect on PDGFR-β protein and VEGFR-2 protein is significantly better than HIF-S. Cyclo-A7R-RRR-HIF-S also has a good degradation effect on the target protein in U87 cells.

[0156] Furthermore, the use of enzyme-sensitive linkers can significantly improve the degradation effect of HIF-S on target proteins. Specifically, in U251 cells, Cyclo-A7R-RRR-GFLG-HIF-S has a better degradation effect on target proteins than Cyclo-A7R-RRR-HIF-S. For example, when the concentration is 5 μmol / L, the degradation effect of Cyclo-A7R-RRR-GFLG-HIF-S on EphB4 protein in U251 cells is 2.38 times that of Cyclo-A7R-RRR-HIF-S. These results show that the use of enzyme-sensitive linkers can improve the degradation activity of HIF-S on target proteins, further confirming the rationality of the design of enzymatic PDC-PROTAC.

[0157] 8. Cell apoptosis detection

[0158] 1. Cell-penetrating peptide-modified PDC-type PROTAC cell apoptosis detection

[0159] U251 cells and U87 cells in good growth state and logarithmic growth phase were cultured at 10 per well. 6 The cells were inoculated into 6-well cell culture plates at a rate of 10 cells / well. Three wells were set for each sample, and blank wells (no drug) and negative control (HIF-S) were set. After inoculation, the culture plates containing cells were placed at 37°C and 5% CO 2in a constant temperature incubator for 24 hours. HIF-S, CPP-HIF-S and CPP-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution. After 48 hours of administration, the cells were collected and washed with pre-cooled PBS. 100 μL of 1× Combine buffer was added to each tube to resuspend the cells, and then 5 μL of Annexin V-FITC was added and incubated 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 test tube to resuspend the cells, and cell apoptosis was detected by flow cytometry (NovoCyte, CytoFLEX).

[0160] The results of cell apoptosis detection of PDC-type PROTAC modified with cell-penetrating peptides are as follows Figures 17 to 19 As shown, it can be seen that after treatment with CPP-HIF-S and CPP-GFLG-HIF-S, the percentage of membrane-associated protein FITC / PI-positive apoptotic U251 cells increased with the increase of the administration concentration. At low concentrations (administration concentrations of 0.1 μmol / L, 0.5 μmol / L and 1 μmol / L), the difference between CPP-HIF-S and CPP-GFLG-HIF-S in promoting apoptosis of U251 cells was small. At high concentrations (administration concentrations of 10 μmol / L and 30 μmol / L), the pro-apoptotic effect of CPP-GFLG-HIF-S on U251 cells was significantly better than that of CPP-HIF-S, and when the administration concentration of CPP-GFLG-HIF-S was 30 μmol / L, it could induce 24.95% of U251 cells to undergo apoptosis. This indicates that the introduction of enzymatic Linker can increase the release of HIF-S in U251 cells and promote apoptosis of U251 cells. In addition, CPP-GFLG-HIF-S has a strong pro-apoptotic effect on U87 cells, and at high concentrations, its apoptosis-inducing effect on U87 cells is significantly stronger than HIF-S. When the concentration was 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 of the HIF-S group.

[0161] 2. Detection of apoptosis of tumor blood vessels and tumor cells by PDC-type PROTAC modified with cell-penetrating peptides

[0162] U251 cells and U87 cells in good growth state and logarithmic growth phase were cultured at 10 per well. 6 The cells were inoculated into 6-well cell culture plates at a rate of 10 cells / well. Three wells were set for each sample, and blank wells (no drug) and negative control (HIF-S) were set. After inoculation, the culture plates containing cells were placed at 37°C and 5% CO 2in a constant temperature incubator for 24 hours. HIF-S, Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution. 48 hours after administration, the cells were collected and washed with pre-cooled PBS. 100 μL of 1× Combine buffer was added to each tube to resuspend the cells, and then 5 μL of Annexin V-FITC was added and incubated 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 test tube to resuspend the cells, and cell apoptosis was detected by flow cytometry (NovoCyte, Cyto FLEX).

[0163] The results of apoptosis detection of PDC-type PROTAC modified with cell-penetrating peptide targeting tumor blood vessels and tumor cells are as follows Figure 20 to Figure 23 As shown, it can be seen that after treatment with Cyclo-A7R-RRR-HIF-S and Cyclo-A7R-RRR-GFLG-HIF-S, the percentage of membrane-associated protein FITC / PI-positive apoptotic U251 cells increased with the increase of the administration concentration. Cyclo-A7R-RRR-HIF-S has a weaker ability to induce apoptosis of U251 cells. At the same concentration, its pro-apoptotic effect on U251 cells is significantly weaker than that of Cyclo-A7R-RRR-GFLG-HIF-S, indicating that the introduction of enzymatic Linker can increase the release of HIF-S in U251 cells and promote the apoptosis of U251 cells. In addition, Cyclo-A7R-RRR-GFLG-HIF-S has a stronger pro-apoptotic effect on U87 cells. When the concentration was 30 μmol / L, the percentage of membrane-associated protein FITC / PI-positive apoptotic cells in the Cyclo-A7R-RRR-GFLG-HIF-S group was 11.92 times that of the HIF-S group.

[0164] IX. Cell cycle detection

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

[0166] U251 cells in optimal growth conditions and logarithmic growth phase were cultured at 10 6 The cells were inoculated into 6-well cell culture plates at a rate of 10 cells / well. Three wells were set for each sample, and blank wells (no drug) and negative control (HIF-S) were set. After inoculation, the culture plates containing cells were placed at 37°C and 5% CO 2in a constant temperature incubator for 24 hours. HIF-S and CPP-GFLG-HIF-S were selected and prepared into a certain concentration gradient solution. After 48 hours of administration, the cells were collected by centrifugation at 800g for 5 minutes. The supernatant was carefully removed and pre-cooled PBS was added to gently resuspend the cells. The cells were collected by centrifugation again. Pre-cooled 70% ethanol was added to fix the cells at 4°C for 24 hours. The cells were collected and washed with pre-cooled 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, the 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] The results of cell cycle detection of PDC-type PROTAC modified with cell-penetrating peptides are as follows Figure 24 to Figure 25 As shown, it can be seen that compared with HIF-S, the application of CPP-GFLG-HIF-S blocks U251 cells in the S phase, and the proportion of cells arrested in the S phase increases with the increase of the administration concentration. When the concentration is 10μmol / L, CPP-GFLG-HIF-S can block 58.87% of U251 cells in the S phase.

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

[0169] U251 cells in optimal growth conditions and logarithmic growth phase were cultured at 10 6 The cells were inoculated into 6-well cell culture plates at a rate of 10 cells / well. Three wells were set for each sample, and blank wells (no drug) and negative control (HIF-S) were set. After inoculation, the culture plates containing cells were placed at 37°C and 5% CO 2 in a constant temperature incubator for 24 hours. Cyclo-A7R-RRR-GFLG-HIF-S was selected and prepared into a certain concentration gradient solution. After 48 hours of administration, the cells were collected by centrifugation at 800g for 5 minutes. The supernatant was carefully removed and pre-cooled PBS was added to gently resuspend the cells. The cells were collected by centrifugation again. Pre-cooled 70% ethanol was added to fix the cells at 4°C for 24 hours. The cells were collected and washed with pre-cooled 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, the 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] The results of cell cycle detection of PDC-type PROTAC modified with cell-penetrating peptide targeting tumor blood vessels and tumor cells are as follows Fig.26 As shown, it can be seen that the application of Cyclo-A7R-RRR-GFLG-HIF-S blocks U251 cells in the S phase, and the proportion of cells arrested in the S phase increases with the increase of the administration concentration. When the concentration is 10μmol / L, Cyclo-A7R-RRR-GFLG-HIF-S can block 51.52% of U251 cells in the S phase.

[0171] In summary, the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides and the enzyme-sensitive PDC-type PROTAC modified with tumor vascular and tumor cell-targeted penetrating peptides can be used to prepare anti-tumor drugs (targeted protein degradation and membrane permeation), have certain human brain glioma cell targeting and membrane permeation capabilities, and have little effect on the cell viability of U251 cells, U87 cells and HEK293 cells, have proliferation inhibitory activity on U251 cells and U87 cells, can degrade target proteins in U251 cells and U87 cells, can induce apoptosis of U251 cells and U87 cells, and affect the cell cycle of U251 cells. The modification of cell-penetrating peptides and enzyme-sensitive linkers, as well as the modification of tumor vascular and tumor cell-targeted penetrating peptides and enzyme-sensitive linkers can expand the structural diversity of PROTAC. At the same time, activity experiments show that the introduction of cell-penetrating peptides and enzyme-sensitive linkers or tumor vascular and tumor cell-targeted 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 serve as another important area for PROTAC development.

[0172] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, characterized in that: It is obtained by modifying PROTAC with cell-penetrating peptides; Wherein, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO.10 or SEQ ID NO.11, and the target protein ligand in the PROTAC is an anti-tumor drug.

2. The cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 1, characterized in that The amino acid sequence of PROTAC is shown in SEQ ID NO.

1.

3. The cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 1 or 2, characterized in that It also includes enzyme-sensitive linkers, cell-penetrating peptides and enzyme-sensitive linkers that co-modify PROTACs; Wherein, the amino acid sequence of the enzyme-sensitive Linker is GFLG.

4. The cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 3, characterized in that When a cell-penetrating peptide is used alone for modification, the cell-penetrating peptide and PROTAC are linked by a covalent bond; when a cell-penetrating peptide and an enzyme-sensitive linker are used for co-modification, the cell-penetrating peptide, the enzyme-sensitive linker and PROTAC are linked by a covalent bond.

5. The method for synthesizing the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide according to any one of claims 1 to 4, characterized in that: An enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide was prepared through an artificial solid-phase synthesis method based on FMOC amino acids.

6. Use of the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide according to any one of claims 1 to 4 in the preparation of an anti-tumor drug preparation.

7. The use according to claim 6, characterized in that: The anti-tumor drug is an anti-glioma drug.

8. The use according to claim 7, characterized in that: The anti-glioma drug is a drug targeting human brain glioma cells.

9. The use according to claim 6, characterized in that: The concentration of the enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide was 1.0×10 -1 ~100.0μmol / L.

10. An anti-glioma composition, characterized in that: It comprises other active ingredients with anti-glioma effects, the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide according to any one of claims 1 to 4, and one or more pharmaceutically acceptable carriers or excipients.

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