A human leukemia cell-targeting penetrating peptide-modified enzyme-sensitive PDC-type PROTAC as well as a preparation method and application thereof

Abstract

This invention discloses an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeting transmembrane peptide, its preparation method, and its applications, belonging to the field of tumor targeted therapy technology. This PDC-type PROTAC is composed of a hypoxia-inducible factor 1α (HIF-1α) protein fragment linked to imatinib via dodecanoic acid, and further modified with the human leukemia cell-targeting transmembrane peptide Cyclo-C9C-R and the cathepsin B-sensitive sequence GFLG. The modified PDC-type PROTAC can release the original drug under the catalysis of cathepsin B, with minimal impact on THP1 cell viability, but effectively inhibits the proliferation of K562 and KU812 cells, degrades target proteins, induces apoptosis, and affects the cell cycle. This gives the PDC-type PROTAC significant advantages in the preparation of anti-tumor drugs, particularly showing promising application prospects in the development of drugs targeting human chronic myeloid leukemia cells and human peripheral blood basophilic leukemia cells, opening up a new field for PROTAC drug development.

Description

Technical Field

[0001] This invention belongs to the field of tumor targeted therapy technology, specifically relating to an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeting transmembrane peptide, its preparation method, and its application. Background Technology

[0002] Leukemia is a disease caused by malignant changes in white blood cells or white blood cell precursor cells, and it is one of the major malignant tumors threatening human health. Radiation exposure, certain types of chemotherapy, or exposure to certain chemicals can increase the risk of developing leukemia. One cause of this disease is chromosomal rearrangement, where abnormal chromosomes interfere with normal cell division. Clinically, it presents with varying degrees of anemia, fever, bleeding, as well as hepatosplenomegaly, lymphadenopathy, and joint pain. Targeted drug therapy strategies can improve the efficiency of leukemia cell-targeted therapy. Proteolysis-targeting chimeras (PROTACs) are a novel drug design strategy that utilizes the intracellular ubiquitin-proteasome system to selectively degrade target proteins. PROTACs can not only degrade target proteins but also target targets traditionally considered difficult to drug. PROTAC technology has shown great potential in leukemia treatment.

[0003] While PROTAC technology has shown great potential in treating malignant tumors such as leukemia, compared with traditional small molecules, PROTACs have larger molecular weights, greater flexibility, more hydrogen bond donors, and larger polar surface areas. These factors lead to poorer physicochemical properties such as solubility, stability, and metabolic characteristics, as well as lower cell permeability and oral bioavailability. Therefore, it is essential to develop new PROTAC types to improve their biological activity and therapeutic efficacy. Peptide-drug conjugates (PDCs) are an emerging targeted therapy approach. A PDC is a conjugate consisting of a peptide, a linker, and a cytotoxic payload. It aims to leverage the high membrane permeability and targeting ability of peptides, as well as the protein degradation capabilities of PROTACs, to achieve more precise and efficient disease treatment. Although PDCs have shown great potential in the preparation of anti-leukemia drugs, some technical limitations also exist. For example, peptides have poor stability in vivo and are easily degraded by proteases, resulting in a short circulation time of PDCs in vivo and difficulty in fully penetrating into tumor tissues. Tissue penetration is limited, as the penetration depth of PDCs is limited by the molecular weight and physicochemical properties of the peptides, making it difficult for PDCs to reach the core of tumor tissues and affecting their killing effect on tumor cells. Oral administration is inefficient, as peptides are easily degraded by digestive enzymes in the gastrointestinal tract and are easily affected by enterohepatic circulation and first-pass elimination effects, resulting in extremely low bioavailability of PDCs when administered orally. Currently marketed and investigational PDC drugs all require injection administration, which limits the flexibility of their administration routes and patient acceptance. Summary of the Invention

[0004] The shortcomings of existing PROTAC and PDC drugs in terms of solubility, stability, penetration, and oral bioavailability limit their application in antitumor therapy. Furthermore, there are no reported technologies to improve the membrane permeability and bioactivity of PDC-type PROTACs through human leukemia cell-targeting transmembrane peptides and enzymatic linkers. The purpose of this invention is to provide an enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptides, its preparation method, and its applications.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention discloses an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeting transmembrane peptide, wherein the enzyme-sensitive PDC-type PROTAC is obtained by modifying a PROTAC with a human leukemia cell-targeting transmembrane peptide; and the target protein ligand in the PROTAC is an anti-leukemia drug.

[0007] The human leukemia cell-targeting transmembrane peptide sequence is shown in SEQ ID NO.4;

[0008] The sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.2.

[0009] The enzyme-sensitive PDC-type PROTAC also includes an enzyme-sensitive sequence, which is obtained by modifying PROTAC with human leukemia cell-targeting membrane-penetrating peptide and an enzyme-sensitive sequence. The enzyme-sensitive sequence is a cathepsin B-sensitive sequence.

[0010] Preferably, the sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.3.

[0011] Preferably, the anti-leukemia drug is imatinib.

[0012] Preferably, the human leukemia cell-targeting membrane-penetrating peptide is Cyclo-C9C-R.

[0013] Preferably, the amino acid sequence of the enzyme-sensitive sequence is as shown in SEQ ID NO.1.

[0014] When modified with cell-penetrating peptide alone, human leukemia cell-targeting cell-penetrating peptide is covalently linked to PROTAC;

[0015] When modified with human leukemia cell-targeting transmembrane peptide and enzyme-sensitive sequence, the human leukemia cell-targeting transmembrane peptide, enzyme-sensitive sequence and PROTAC are covalently linked.

[0016] The PROTAC is obtained by linking a hypoxia-inducible factor-1α protein fragment with a drug molecule via dodecanoic acid. The sequence of the hypoxia-inducible factor-1α protein fragment is Leu-Ala-Pro(OH)-Tyr-Ile (as shown in SEQ ID NO.5).

[0017] In a second aspect, the present invention discloses a method for preparing enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides targeting human leukemia cells. The enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides is prepared by an artificial solid-phase synthesis method based on FMOC amino acids.

[0018] In a third aspect, the present invention discloses the application of the enzyme-sensitive PDC-type PROTAC modified with the above-mentioned human leukemia cell-targeting transmembrane peptide in the preparation of anti-leukemia drugs.

[0019] Preferably, the anti-leukemia drug is a drug that targets human chronic myeloid leukemia cells or human peripheral blood basophilic leukemia cells.

[0020] In a fourth aspect, the present invention discloses an anti-leukemia composition comprising other active ingredients having anti-leukemia activity, the above-mentioned human leukemia cell-targeting transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC, and a pharmaceutically acceptable carrier.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeting transmembrane peptide. For the first time, transmembrane peptides and / or enzymatically digestible linkers are introduced into the design of PROTACs, proposing a novel leukemia treatment strategy. This not only solves the problems of poor solubility, stability, and cell permeability of existing PROTACs but also improves the targeting ability of PROTACs. The human leukemia cell-targeting transmembrane peptide can specifically recognize and bind to receptors or antigens on the surface of leukemia cells, guiding the PROTAC into the target cells. The enzyme-sensitive sequence is cleaved under the action of specific intracellular enzymes, releasing the PDC active ingredient, achieving targeted drug delivery and controlled release.

[0023] The preparation method provided by this invention employs a solid-phase synthesis method using FMOC amino acids to prepare enzyme-sensitive PDC-type PROTACs modified with cell-penetrating peptides targeting human leukemia cells. This method is efficient, precise, and flexible, capable of preparing high-purity and highly active PROTAC drugs, providing new ideas and methods for the development of anti-tumor drugs. By combining cell-penetrating peptides, enzymatic linkers, and artificial solid-phase synthesis, the solubility, stability, cell permeability, and bioactivity of PROTACs are significantly improved. This method also has the advantages of low cost and high yield, and has broad application prospects and important research value.

[0024] The application provided by this invention, by using PDC-type PROTAC in the preparation of anti-leukemia drugs, can develop novel anti-leukemia drugs with higher targeting and lower toxicity. By introducing human leukemia cell-targeting membrane-penetrating peptides and / or enzymatically hydrolyzed linkers, not only is the solubility, stability, and cell permeability of PROTAC improved, but its biological activity and therapeutic effect are also enhanced, opening up new avenues for its application in anti-leukemia treatment, with broad application prospects and important clinical significance.

[0025] The anti-leukemia composition provided by this invention can further broaden its therapeutic scope and improve its therapeutic effect by combining PDC-type PROTAC with other active ingredients with anti-leukemia activity. The addition of pharmaceutically acceptable carriers or excipients also helps to improve the stability and bioavailability of the composition. This anti-leukemia composition has multiple active ingredients that can exert a synergistic effect to improve the therapeutic effect of leukemia. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method for synthesizing enzyme-sensitive PDC-type PROTAC modified with leukemia cell-targeted transmembrane peptides, as described in this invention.

[0027] Figure 2 The MALDI-TOF-MS spectrum of HIF-IMA;

[0028] Figure 3 MALDI-TOF-MS spectrum of Cyclo-C9C-R-HIF-IMA;

[0029] Figure 4 MALDI-TOF-MS spectrum of Cyclo-C9C-R-GFLG-HIF-IMA;

[0030] Figure 5 This is a diagram showing the release process of enzyme-sensitive PDC-type PROTAC modified with a membrane-penetrating peptide targeting leukemia cells (under enzyme presence conditions).

[0031] Figure 6 This is a graph showing the effect of the PDC-type PROTAC modified with leukemia cell-targeting membrane-penetrating peptide on the viability of THP1 cells.

[0032] Figure 7 This is a graph showing the inhibitory activity of the PDC-type PROTAC modified with leukemia cell-targeting membrane-penetrating peptide against the proliferation of K562 cells.

[0033] Figure 8 This is a graph showing the inhibitory activity of the PDC-type PROTAC modified with leukemia cell-targeting membrane-penetrating peptide against the proliferation of KU812 cells.

[0034] Figure 9 This image shows the degradation effect of the PDC-type PROTAC modified with leukemia cell-targeting transmembrane peptide on target proteins in K562 cells. In the image, A represents the degradation effect of HIF-IMA on target proteins in K562 cells, C represents the degradation effect of Cyclo-C9C-R-HIF-IMA on target proteins in K562 cells, E represents the degradation effect of Cyclo-C9C-R-GFLG-HIF-IMA on target proteins in K562 cells, and B, D, and F represent the semi-quantitative results of target protein expression levels in A, C, and E.

[0035] Figure 10The results show the degradation effects of PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptides on intracellular target proteins in KU812 cells. Among them, A represents the degradation effect of HIF-IMA on intracellular target proteins in KU812 cells, C represents the degradation effect of Cyclo-C9C-R-HIF-IMA on intracellular target proteins in KU812 cells, E represents the degradation effect of Cyclo-C9C-R-GFLG-HIF-IMA on intracellular target proteins in KU812 cells, and B, D, and F represent the semi-quantitative results of target protein expression levels in A, C, and E, respectively.

[0036] Figure 11 This invention relates to the effect of enzyme-sensitive PDC-type PROTAC modified with a membrane-penetrating peptide targeting leukemia cells on apoptosis of K562 cells; wherein, A represents the effect of HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on apoptosis of K562 cells, and B represents the quantitative results of apoptotic cells in A.

[0037] Figure 12 This invention relates to the effect of enzyme-sensitive PDC-type PROTAC modified with a membrane-penetrating peptide targeting leukemia cells on apoptosis of KU812 cells; wherein, A represents the effect of HIF-IMA, Cyclo-C9C-R-HIF-IMA, and Cyclo-C9C-R-GFLG-HIF-IMA on apoptosis of KU812 cells; and B represents the quantitative results of apoptotic cells in A.

[0038] Figure 13 The effect of PDC-type PROTAC on the K562 cell cycle; where A represents the effect of HIF-IMA on the K562 cell cycle; B represents the quantitative results of A.

[0039] Figure 14 The invention relates to the effect of enzyme-sensitive PDC-type PROTAC modified with a membrane-penetrating peptide targeting leukemia cells on the cell cycle of K562 cells; wherein, A is the effect of Cyclo-C9C-R-GFLG-HIF-IMA on the cell cycle of K562 cells, and B is the quantitative result of A.

[0040] Figure 15 The effect of PDC-type PROTAC on the cell cycle of KU812 cells; where A represents the effect of HIF-IMA on the cell cycle of KU812 cells; and B represents the quantitative results of A.

[0041] Figure 16This invention relates to the effect of enzyme-sensitive PDC-type PROTAC modified with a membrane-penetrating peptide targeting leukemia cells on the cell cycle of KU812 cells; wherein, A represents the effect of Cyclo-C9C-R-GFLG-HIF-IMA on the cell cycle of KU812 cells, and B represents the quantitative result of A. Detailed Implementation

[0042] This invention provides a PDC-type PROTAC modified with human leukemia cell-targeting membrane-penetrating peptide. The PDC-type PROTAC (sequence shown as SEQ ID NO.1 in Table 1) is obtained by modifying the human leukemia cell-targeting membrane-penetrating peptide and enzyme-sensitive sequence. The sequences of the PDC-type PROTAC modified with human leukemia cell-targeting membrane-penetrating peptide are shown as SEQ ID NO.2~SEQ ID NO.3 in Table 1.

[0043] The PROTAC is obtained by linking a hypoxia-inducible factor-1α protein fragment and a drug molecule via dodecanoic acid, and the sequence of the hypoxia-inducible factor-1α protein fragment is LAP(OH)YI (as shown in SEQ ID NO.5).

[0044] Among them, the human leukemia cell-targeting membrane-penetrating peptide is Cyclo-C9C-R (as shown in SEQ ID NO.4), and the enzyme-sensitive sequence is the cathepsin B-sensitive sequence GFLG.

[0045] Table 1: Amino Acid Sequence List

[0046]

[0047] Note: In the amino acid sequence, "L" represents "Leucine", "A" represents "Alanine", "P(OH)" represents "Hydroxy-L-proline", "Y" represents "Tyrosine", "I" represents "Isoleucine", "IMA" represents "Imatinib", "C(....)" represents cyclization, "K" represents "Lysine", "R" represents "Arginine", "F" represents "Phenylalanine", "W" represents "Tryptophan", "S" represents "Serine", "E" represents "Glutamic acid", and "G" represents "Glycine".

[0048] 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.

[0049] 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.

[0050] I. Experimental Materials

[0051] 1. Experimental drugs and reagents: Rink Amide-MBHA resin (Bai Ling Wei), Fmoc protected amino acids, piperidine (Sinopharm Group), N,N-dimethylformamide (DMF, Comio Chemical Reagent), DCM (Comio Chemical Reagent), HATU (Bide Pharmaceutical), HOBt (Bide Pharmaceutical), DIPEA (Bide Pharmaceutical), (benzotriazol-1-yloxy)tripyrrolidinyl hexafluorophosphate (PyBop, Bide Pharmaceutical), triethylamine (TFA, Aladdin), TIS (Aladdin), 1,3-dimethoxybenzene (Aladdin), DODT (Aladdin), chromatographic acetonitrile (Aladdin), PBS buffer (CORNING), RPMI 1640 (CORNING), penicillin-streptomycin solution (double antibody) (MCE), fetal bovine serum (NEWZERUM), Trypsin (CORNING), DMSO (MP Biomedicals), MTT (Solarbio).

[0052] 2. Cells used in the experiment: K562 (human chronic myeloid leukemia cells), KU812 (human peripheral blood basophilic leukemia cells) and THP1 (human leukemia mononuclear cell line).

[0053] II. Synthetic enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptides

[0054] like Figure 1 As shown, enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptides was synthesized using a solid-phase synthesis method based on FMOC amino acids.

[0055] 1. Coupling of N-demethylimatinib with dodecanoic acid

[0056] N - Coupling of demethylimatinib with dodecanoic acid (synthesis of 12-(4-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)carbamoyl)benzyl)piperazin-1-yl)-12-oxododecanoic acid)

[0057] Dodecanoic acid (0.45 g, 1.95 mmol) and PyBop (1.02 g, 1.95 mmol) were dissolved in anhydrous DMF, and TEA (0.98 g, 9.75 mmol) was added dropwise to the solution; [The remaining text appears to be incomplete and requires further context.] N - Anhydrous DMF solution of desmethylimatinib (0.47 g, 0.98 mmol) was prepared, and the system pH was monitored to approximately 8. The reaction was carried out at room temperature for 4 hours. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate (3×). The combined organic phases were washed with brine, dried over Na2SO4, and evaporated under reduced pressure to give the crude product. The crude product was separated by column chromatography to obtain a grayish-white solid (0.55 g, 82% yield). Mass spectrometry (ESI, m / z): 692.35 [M + H]. + 690.30 [M - H] - ,get N - The coupling product of demethylimatinib and dodecanoic acid: 12-(4-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)carbamoyl)benzyl)piperazin-1-yl)-12-oxododecanoic acid.

[0058] 2. Synthesis of Cyclo-C9C-R-GFLG-HIF-IMA

[0059] (1) Resin preparation and swelling

[0060] Take Rink Amide-MBHA resin (50.0 mg, 0.32 mmol / g, 0.016 mmol) and place it in a reaction vessel (centrifuge column). Add 5 mL of DMF / DCM (1:1) mixed solvent and allow the resin to swell fully for 45 minutes. Then, filter under reduced pressure and discard the filtrate.

[0061] (2) Deprotection reaction

[0062] Add 20% piperidine / DMF mixture and carry out the FMOC deprotection reaction for 10 minutes. Filter the filtrate under reduced pressure and repeat the above operation twice to completely remove the Fmoc protecting group at the N end of the Rink Amide-MBHA resin. After filtration, thoroughly wash the resin with DCM / DMF (1:1) mixture to remove residual piperidine and DMF.

[0063] (3) Amino acid linkage

[0064] Weigh out Fmoc-Glu-Oall (32.8 mg, 0.08 mmol), HATU (30.3 mg, 0.08 mmol), HOBt (12.2 mg, 0.08 mmol), and DIPEA (37.6 μL, 0.08 mmol) and dissolve them in an appropriate amount of DMF to obtain a mixture. Add the mixture to a centrifuge column and react for 45 minutes to successfully ligate Fmoc-Glu-Oall onto the resin. Filter and thoroughly wash the resin with DCM / DMF (1:1) to remove unreacted amino acids and reagents. Ligate amino acids sequentially according to the sequences shown in SEQ ID NO.1 to SEQ ID NO.3 in Table 1. Repeat the above ligation and washing steps.

[0065] (4) Cyclization reaction

[0066] For peptides requiring cyclization, after the deprotection reaction was completed, 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 ensure the complete cyclization reaction.

[0067] (5) Coupling product linkage

[0068] After completing the synthesis of the polypeptide portion, N - The conjugate of demethylimatinib and dodecanoic acid (12-(4-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)carbamoyl)benzyl)piperazin-1-yl)-12-oxododecanoic acid) (69.1 mg, 0.10 mmol) and the conjugate DIC (12.4 μL, 0.08 mmol) were added to a centrifuge column and reacted overnight on a rotary mixer to allow the conjugate to be successfully linked to the polypeptide chain, forming the complete Cyclo-C9C-R-GFLG-HIF-IMA molecule;

[0069] (6) Product pyrolysis and purification

[0070] After synthesis, it is processed using a cleavage reagent (V TFA V TIS V 1,3-二甲氧基苯 V DODT V H2OThe product was lysed from the resin using a lysis reagent (90:2.5:2.5:2.5:2.5). The lysis reagent broke the bonds between the resin and the polypeptide chain, releasing the product. DCM was added to the centrifuge column to thoroughly wash the product and collect the liquid. The product was dissolved and residual lysis reagent was removed. The solvent was evaporated, and ice-cold ether was added to the centrifuge tube. The product precipitated, 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, and the product was gently washed. The product was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded to obtain the crude peptide. The crude peptide was stored at -80°C for subsequent purification.

[0071] (7) Purification and characterization

[0072] See appendix Figures 2-4 The crude peptides were purified by linear gradient elution using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) on a Shimadzu LC-2030. Purity analysis was performed using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm) on a Shimadzu LC-2030C 3D Plus equipped with a PDA detector. Characterization was performed using a Hexin CMI-1600 MALDI-TOF-MS instrument, with the purity of the purified peptides detected at a wavelength of 254 nm to ensure a purity >90.0%.

[0073] Analysis of MALDI-TOF-MS spectra confirmed the correct synthesis, purity, and structural characteristics of HIF-IMA, Cyclo-C9C-R-HIF-IMA, and Cyclo-C9C-R-GFLG-HIF-IMA molecules, providing a reliable foundation for subsequent biological research and applications.

[0074] III. Detection of the release of enzyme-sensitive PDC-type PROTAC modified with targeted transmembrane peptides from human leukemia cells

[0075] The release of PDC-PROTAC from enzyme-sensitive PDC-PROTAC modified with human leukemia cell-targeting transmembrane peptides was detected using high-performance liquid chromatography (HPLC). The specific method is as follows:

[0076] A reaction solution of Cyclo-C9C-R-GFLG-HIF-IMA was prepared at a concentration of 1 mg / mL. 5 μL of cathepsin B (C-6×His, 1.39 mg / mL, MedChemExpress) was added to each solution, while 5 μL of PBS was added to the control group. The reaction system was 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). The changes in peak height and peak area of ​​Cyclo-C9C-R-GFLG-HIF-IMA / HIF-IMA were observed. Chromatographic conditions: Shimadzu C18 column (C18-120 / 5μm, 10.0×250 mm), mobile phase was acetonitrile containing 0.05% TFA and water containing 0.05% TFA, eluted with a gradient of 20%-90% acetonitrile, flow rate: 1 ml / min, column temperature: 30℃, detection volume: 50 μL, detection wavelength: 254 nm.

[0077] Release of PDC-PROTAC from enzyme-sensitive PDC-PROTAC modified with transmembrane peptides targeting human leukemia cells, such as Figure 5 As shown.

[0078] from Figure 5 As can be seen, Cyclo-C9C-R-GFLG-HIF-IMA (sequence shown as SEQ ID NO.3 in Table 1) achieved rapid release of HIF-IMA under the catalysis of cathepsin B, with a release rate of 46.22% within 1 hour, over 90% release within 4 hours, and 98.74% release within 6 hours. These results indicate that linking the human leukemia cell-targeting transmembrane peptide to PDC-PROTAC via an enzyme-sensitive linker can achieve sufficient release of PDC-PROTAC.

[0079] IV. Detection of PDC-type PROTAC cytotoxicity modified with targeted transmembrane peptides in human leukemia cells

[0080] The cytotoxicity of PDC-type PROTAC modified with a human leukemia cell-targeting transmembrane peptide against tumor cells and control cells was determined using the MTT assay. The specific method is as follows:

[0081] 1. Cell seeding: K562 cells, KU812 cells, and THP1 cells in the growth exponential phase were diluted to 10⁻⁶ cells / mL using RPMI 1640 medium. 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℃ and 5% CO2 for 12 h.

[0082] 2. Drug administration: 20 μL of different concentrations of HIF-IMA, Cyclo-C9C-R-HIF-IMA, or Cyclo-C9C-R-GFLG-HIF-IMA were added to each well, resulting in a final concentration of the compound in the well: 3.0 × 10⁻⁶. -1 μmol / L, 1.0 μmol / L, 3.0 μmol / L, 10.0 μmol / L, 50.0 μmol / L, 100.0 μmol / L, with 3 replicates for each concentration and 6 replicates for the negative control. The mixture was incubated at 37℃ and 5% CO2 for 48 h.

[0083] 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.

[0084] The formula for calculating the cell inhibition rate is:

[0085] Inhibition rate % = (Average OD value of control wells - Average OD value of drug-treated group) / Average OD value of control wells × 100%;

[0086] 4. Experimental Results: From Figure 6 As can be seen from the data, the cytotoxicity analysis showed that 3.0 × 10 -1 Within a concentration range of μmol / L to 100.0 μmol / L, compounds HIF-IMA, Cyclo-C9C-R-HIF-IMA, or Cyclo-C9C-R-GFLG-HIF-IMA had minimal impact on the cell viability of K562, KU812, and THP1 cells, with cell survival rates all exceeding 80%. This demonstrates that these PDC-type PROTACs modified with transmembrane peptides targeting human leukemia cells have a certain degree of safety at the cellular level.

[0087] V. Detection of the inhibitory activity of PDC-type PROTAC tumor cells modified with human leukemia cell-targeting transmembrane peptides on proliferation

[0088] K562 and KU812 cells in good growth condition and logarithmic growth phase were centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in RPMI 1640 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 and negative controls. After seeding, the cell-containing culture plates were incubated at 37°C and 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA, or Cyclo-C9C-R-GFLG-HIF-IMA were prepared as concentration gradient solutions, and administered to K562 and KU812 cells at a dose of 20 μL per well. Forty-eight hours after administration, MTT solution was added, and the cells were further cultured at 37°C and 5% CO2 for 4 hours. Cell inhibition rate was calculated after measuring absorbance.

[0089] The formula for calculating the cell inhibition rate is:

[0090] Inhibition rate % = (Average OD value of control wells - Average OD value of drug-treated group) / Average OD value of control wells × 100%;

[0091] The inhibitory activity of PDC-type PROTAC tumor cells modified with transmembrane peptides targeting human leukemia cells, such as Figure 7 and Figure 8 As shown.

[0092] from Figure 7 and Figure 8 It can be seen that Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA exhibit superior inhibitory activity against the proliferation of K562 and KU812 cells compared to HIF-IMA, and their inhibitory activity increases with increasing concentration. At low concentrations (0.3 μmol / L and 1.0 μmol / L), Cyclo-C9C-R-HIF-IMA showed significantly higher inhibitory activity against the proliferation of K562 cells than HIF-IMA, increasing the inhibitory activity of HIF-IMA by 5.67-fold and 7.01-fold, respectively. These results indicate that the introduction of human leukemia cell-targeting transmembrane peptides can significantly enhance the inhibitory activity of HIF-IMA against the proliferation of K562 cells. Furthermore, the introduction of the enzyme-sensitive linker (GFLG) also promotes the antiproliferative activity of HIF-IMA, and this effect is applicable at all concentrations. These results further demonstrate the rationality of the PDC-type PROTAC design modified with human leukemia cell-targeting transmembrane peptides.

[0093] VI. Detection of degradation activity of PDC-type PROTAC protein modified with targeted transmembrane peptides from human leukemia cells

[0094] K562 and KU812 cells in good growth condition and 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 set up for each sample, along with blank wells and negative controls. After seeding, the cell-containing culture plates were incubated at 37°C with 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA, Cyclo-C9C-R-GFLG-HIF-IMA, and imatinib were selected and prepared into concentration gradient solutions. Forty-eight hours after administration, protein samples were collected, and Western blot analysis was performed to investigate the protein degradation activity under different administration methods.

[0095] Results of PDC-type PROTAC protein degradation activity targeting transmembrane peptide modification in human leukemia cells are as follows: Figures 9 to 10 As shown.

[0096] from Figure 9 and Figure 10 As can be seen, HIF-IMA exhibits poor degradation activity against target proteins in K562 and KU812 cells. At a concentration of 30 μmol / L, it only degrades 22.55% of BCR-ABL protein in K562 cells, and has no significant effect on downstream STAT5 phosphorylation. In KU812 cells, even at a concentration of 50 μmol / L, it only degrades 20.01% of BCR-ABL protein. However, after modification with human leukemia cell-targeting transmembrane peptides, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA show significantly enhanced degradation activity against target proteins in both K562 and KU812 cells. In K562 cells, at a concentration of 30 μmol / L, Cyclo-C9C-R-HIF-IMA exhibited slightly higher degradation activity against BCR-ABL protein than HIF-IMA, degrading 28.66% of the BCR-ABL protein. However, at a concentration of 50 μmol / L, the degradation rate of BCR-ABL protein in K562 cells was 1.46 times that of HIF-IMA, indicating that the introduction of Cyclo-C9C-R can enhance the protein degradation activity of HIF-IMA. Furthermore, Cyclo-C9C-R-HIF-IMA also showed superior degradation activity against target proteins in KU812 cells compared to HIF-IMA, not only enhancing the degradation of BCR-ABL protein by HIF-IMA but also improving the inhibition of downstream STAT5 phosphorylation.

[0097] Furthermore, the use of enzyme-sensitive linkers significantly enhances the degradation of target proteins by HIF-IMA, even at low concentrations. Specifically, in K562 cells, when the concentration of Cyclo-C9C-R-GFLG-HIF-IMA was 0.3 μmol / L, compared to Cyclo-C9C-R-HIF-IMA, the degradation rate of BCR-ABL protein was increased by 1.79 times, and it also promoted the inhibition of downstream STAT5 phosphorylation. In KU812 cells, Cyclo-C9C-R-GFLG-HIF-IMA significantly enhanced the inhibition of STAT5 phosphorylation, a downstream protein of BCR-ABL. These results further demonstrate that the introduction of enzyme-sensitive linkers can improve the degradation activity of HIF-IMA on target proteins, confirming the applicability of this strategy to hematologic malignancies. The investigation into the protein degradation mechanism of HIF-IMA showed that the proteasome inhibitor MG-132 could block protein degradation induced by Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA, with a more significant blocking effect on protein degradation induced by Cyclo-C9C-R-GFLG-HIF-IMA, indicating that it works through the proteasome pathway.

[0098] VII. Apoptosis Detection of PDC-type PROTAC Cells Modified with Targeted Transmembrane-Penetrating Peptides in Human Leukemia Cells

[0099] K562 and KU812 cells in good growth condition and 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, along with blank wells and a negative control. After seeding, the cell-containing plates were incubated at 37°C with 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA, and Cyclo-C9C-R-GFLG-HIF-IMA were selected and prepared as concentration gradient solutions. Forty-eight hours after 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).

[0100] Apoptosis detection results of PDC-type PROTAC cells modified with targeted transmembrane peptides in human leukemia cells are as follows: Figure 11 and Figure 12 As shown.

[0101] from Figure 11 and Figure 12 As can be seen, compared with HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA significantly enhanced the ability to induce apoptosis in K562 cells, and showed good effects at both low and high concentrations. At a concentration of 0.3 μmol / L, the percentage of K562 cell apoptosis induced by Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA was 1.57 times and 1.48 times that of HIF-IMA, respectively. At a concentration of 30 μmol / L, the apoptosis-inducing effects of Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on K562 cells were even stronger, being 10.53 times and 12.09 times that of HIF-IMA, respectively. Furthermore, Cyclo-C9C-R-GFLG-HIF-IMA exhibited a stronger apoptosis-inducing effect on K562 cells than Cyclo-C9C-R-HIF-IMA. At a concentration of 10 μmol / L, the apoptosis-inducing effect of Cyclo-C9C-R-GFLG-HIF-IMA was 1.85 times that of Cyclo-C9C-R-HIF-IMA. HIF-IMA, Cyclo-C9C-R-HIF-IMA, and Cyclo-C9C-R-GFLG-HIF-IMA showed weaker apoptosis-inducing effects on KU812 cells, but Cyclo-C9C-R-GFLG-HIF-IMA still possessed the best ability to promote KU812 cell apoptosis. These results indicate that modification with human leukemia cell-targeting transmembrane peptides and enzymatically digestible linkers significantly enhances the apoptosis-promoting effect of HIF-IMA on tumor cells.

[0102] VIII. Cell cycle detection of PDC-type PROTAC cells modified with targeted transmembrane peptides in human leukemia cells

[0103] K562 and KU812 cells, which were in optimal growth conditions and in the logarithmic growth phase, were cultured at 10 cells 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, along with blank wells and a negative control. After seeding, the cell-containing culture plates were incubated at 37°C with 5% CO2 for 24 hours. HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA were selected and prepared as concentration gradient solutions. Forty-eight hours after 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.

[0104] Cell cycle assay results of PDC-type PROTAC cells modified with targeted transmembrane peptides in human leukemia cells are as follows: Figures 13 to 16 As shown.

[0105] from Figures 13 to 16 As can be seen, compared with HIF-IMA, the application of Cyclo-C9C-R-GFLG-HIF-IMA arrested K562 cells in the G1 / M phase. At a concentration of 30 μmol / L, Cyclo-C9C-R-GFLG-HIF-IMA was able to arrest 69.87% of K562 cells in the G1 / M phase, which was higher than the effect of HIF-IMA at the same concentration (59.64%). For KU812 cells, Cyclo-C9C-R-GFLG-HIF-IMA arrested them in the S phase. At a concentration of 30 μmol / L, Cyclo-C9C-R-GFLG-HIF-IMA was able to arrest 64.15% of KU812 cells in the S phase, which was higher than the effect of HIF-IMA at the same concentration (55.05%).

[0106] In summary, enzyme-sensitive PDC-type PROTACs modified with human leukemia cell-targeting transmembrane peptides can be used to prepare anti-leukemia drugs (targeting protein degradation and transmembrane penetration). They possess certain leukemia cell targeting and transmembrane penetration capabilities, and have minimal impact on the cell viability of K562, KU812, and THP1 cells. They exhibit proliferative inhibitory activity against K562 and KU812 cells, degrading target proteins in these cells and inducing apoptosis, thus affecting their cell cycle. Modification with human leukemia cell-targeting transmembrane peptides and enzyme-sensitive sequences can expand the structural diversity of PROTACs. Furthermore, activity assays show that the introduction of these peptides and sequences significantly enhances the protein degradation effect of PDC-PROTACs, increasing their application scenarios and representing another important area for PROTAC development.

[0107] 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 human leukemia cell-targeting transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC, characterized in that, The enzyme-sensitive PDC-type PROTAC is obtained by covalently modifying a PROTAC with a human leukemia cell-targeting membrane-penetrating peptide; the target protein ligand in the PROTAC is imatinib; the PROTAC is obtained by linking a hypoxia-inducible factor-1α protein fragment with an imatinib drug molecule via dodecanoic acid; the human leukemia cell-targeting membrane-penetrating peptide is cyclized KRFYWLRSE; the hypoxia-inducible factor-1α protein fragment is LA-P(OH)-YI, where P(OH) is hydroxy-L-proline; the structural formula of the enzyme-sensitive PDC-type PROTAC is as follows: 。 2. The enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptide according to claim 1, characterized in that, It also contains the cathepsin B-sensitive sequence GFLG, and the human leukemia cell-targeting transmembrane peptide, enzyme-sensitive sequence, and PROTAC are covalently linked; the structural formula of the enzyme-sensitive PDC-type PROTAC is as follows: 。 3. The application of the enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptide as described in claim 1 or 2 in the preparation of anti-leukemia drugs.

4. The application according to claim 3, characterized in that, The anti-leukemia drug is a drug that targets human chronic myeloid leukemia cells or human peripheral blood basophilic leukemia cells.

5. The application according to claim 3, characterized in that, The concentration of enzyme-sensitive PDC-type PROTAC modified with transmembrane peptides targeting human leukemia cells was 3.0 × 10⁻⁶. -1 ~100.0 μmol / L.

6. An anti-leukemia composition, characterized in that, The anti-leukemia composition comprises other active ingredients with anti-leukemia activity, enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeting transmembrane peptide as described in claim 1 or 2, and one or more pharmaceutically acceptable carriers or excipients.

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