Enzyme-sensitive PDC type PROTAC modified by human leukemia cell targeted cell-penetrating peptide and preparation method and application of enzyme-sensitive PDC type PROTAC

Abstract

The invention discloses a human leukemia cell targeted 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 PDC type PROTAC is formed by connecting a hypoxia inducible factor 1 alpha (HIF-1alpha) protein fragment and imatinib through dodecanedioic acid, and is further modified through a human leukemia cell targeted cell-penetrating peptide Cyclo-C9C-R and a cathepsin B sensitive sequence GFLG. The modified PDC type PROTAC can release original drugs under the catalysis of cathepsin B, has small influence on THP1 cell activity, but can effectively inhibit proliferation of K562 cells and KU812 cells, degrade target proteins, induce cell apoptosis and influence cell cycles, so that the PDC type PROTAC has remarkable advantages in the aspect of preparation of antitumor drugs, and has broad application prospects. Particularly, the compound shows a good application prospect in the development of drugs for targeting human chronic myelogenous leukemia cells and human peripheral blood basophilic leukemia cells, and opens up a new field for the development of PROTAC drugs.

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 targeted to human leukemia cells, and a preparation method and application thereof. Background Art

[0002] Leukemia is a disease caused by malignant changes in white blood cells or white blood cell precursors. It is one of the main malignant tumors that endanger humans. Radiation exposure, some types of chemotherapy, or contact with certain chemicals increase the risk of leukemia. One of the causes of this disease is the rearrangement of chromosome segments, and the abnormal chromosomes produced interfere with the normal cell division. Clinically, different degrees of anemia, fever, bleeding, liver, spleen, lymph node enlargement, and joint pain can be seen. The use of drug targeted therapy strategies to treat leukemia can improve the efficiency of targeted therapy of leukemia cells. Protein degradation targeting chimeras (PROTAC) are a new drug design strategy that uses the intracellular ubiquitin-proteasome system to selectively degrade target proteins. PROTAC can not only degrade target proteins, but also target targets that are traditionally considered difficult to drug. In the treatment of leukemia, PROTAC technology has shown great potential.

[0003] Although PROTAC technology has shown great potential in the treatment of malignant tumors such as leukemia, compared with traditional small molecules, PROTAC has a larger molecular weight, higher flexibility, more hydrogen bond donors, and a larger polar surface area. These factors lead to poor physical and chemical properties such as solubility, stability, and metabolic characteristics, as well as low cell permeability and oral bioavailability. Therefore, it is very necessary to develop new types of PROTACs to improve their biological activity and therapeutic effects. Peptide-Drug Conjugate (PDC) is an emerging targeted therapy. PDC is a conjugated drug consisting of a peptide, a linker, and a cytotoxic payload. It aims to utilize the high membrane permeability and targeting of peptides and the protein degradation ability of PROTAC to achieve more accurate and efficient disease treatment. Although the application of PDC in the preparation of anti-leukemia drugs shows great potential, there are also some technical defects. For example, peptides have poor stability in the body and are easily degraded by proteases, resulting in a short circulation time of PDC in the body and difficulty in fully penetrating into tumor tissues; tissue penetration is limited, and the penetration depth of PDC is limited by the molecular weight and physical and chemical properties of the peptides, resulting in difficulty for PDC to reach the core of tumor tissues, affecting its killing effect on tumor cells; oral administration efficiency is low, 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 PDC when administered orally. Currently, all PDC drugs on the market and under development need to be administered by injection, which limits the flexibility of their administration routes and patient acceptance. Summary of the invention

[0004] In view of the deficiencies of existing PROTAC and PDC drugs in solubility, stability, permeability, oral bioavailability, etc., the application of PROTAC and PDC drugs in anti-tumor treatment is limited, and there is no technical status reported on improving the permeability and biological activity of PDC-type PROTAC by human leukemia cell-targeted transmembrane peptides and enzymatic linkers. The object of the present invention is to provide an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeted transmembrane peptide, and a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeting penetrating peptide is disclosed, wherein the enzyme-sensitive PDC-type PROTAC is obtained by modifying a PROTAC with a human leukemia cell-targeting penetrating peptide; the target protein ligand in the PROTAC is an anti-leukemia drug; The human leukemia cell targeting membrane-penetrating peptide sequence is shown in SEQ ID NO.4; The sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.2.

[0006] The enzyme-sensitive PDC-type PROTAC also includes an enzyme-sensitive sequence, which is obtained by modifying PROTAC with a human leukemia cell-targeted transmembrane peptide and an enzyme-sensitive sequence, and the enzyme-sensitive sequence is a cathepsin B-sensitive sequence.

[0007] Preferably, the sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.3.

[0008] Preferably, the anti-leukemia drug is imatinib.

[0009] Preferably, the human leukemia cell targeting transmembrane peptide is Cyclo-C9C-R.

[0010] Preferably, the amino acid sequence of the enzyme-sensitive sequence is shown as SEQ ID NO.1.

[0011] When cell-penetrating peptide modification is used alone, the human leukemia cell-targeting cell-penetrating peptide is covalently linked to PROTAC; When the human leukemia cell targeting transmembrane peptide and the enzyme-sensitive sequence are used for co-modification, the human leukemia cell targeting transmembrane peptide, the enzyme-sensitive sequence and PROTAC are linked by covalent bonds.

[0012] The PROTAC is obtained by connecting a hypoxia-inducible factor-1α protein fragment to a drug molecule through dodecanedioic acid, and the sequence of the hypoxia-inducible factor-1α protein fragment is Leu-Ala-Pro(OH)-Tyr-Ile (as shown in SEQ ID NO.5).

[0013] In the second aspect of the present invention, a method for preparing the above-mentioned human leukemia cell-targeted 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 human leukemia cell-targeted transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC in the preparation of anti-leukemia drugs.

[0015] Preferably, the anti-leukemia drug is a drug targeting human chronic myeloid leukemia cells or human peripheral blood basophilic leukemia cells.

[0016] In a fourth aspect of the present invention, an anti-leukemia composition is disclosed, comprising other active ingredients having anti-leukemia effects, the above-mentioned human leukemia cell-targeting transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC, and a pharmaceutically acceptable carrier.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an enzyme-sensitive PDC-type PROTAC modified with a human leukemia cell-targeted penetrating peptide. For the first time, a cell-penetrating peptide and / or an enzymatic linker are introduced into the design of PROTAC, proposing a new leukemia treatment strategy, which not only solves the problems of poor solubility, stability and cell permeability of existing PROTACs, but also improves the targeting of PROTACs; the human leukemia cell-targeted penetrating peptide can specifically recognize and bind to receptors or antigens on the surface of leukemia cells, guiding PROTAC into target cells; the enzyme-sensitive sequence is broken under the action of specific enzymes in the cell, releasing the PDC active ingredient, thereby achieving targeted delivery and controllable release of the drug.

[0018] The preparation method provided by the present invention adopts the solid phase synthesis method of FMOC amino acids to prepare an enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide targeting human leukemia cells. The method is efficient, accurate and flexible, and can prepare PROTAC drugs with high purity and strong activity, 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 biological activity of PROTAC are significantly improved; the method also has the advantages of low cost and high yield, and has broad application prospects and important research value.

[0019] The application provided by the present invention can develop new anti-leukemia drugs with higher targeting and lower toxic side effects by applying PDC-type PROTAC to the preparation of anti-leukemia drugs. By introducing human leukemia cell-targeted transmembrane peptides and / or enzymatic linkers, not only the solubility, stability and cell permeability of PROTAC are improved, but also its biological activity and therapeutic effect are enhanced, thus opening up a new path for its application in anti-leukemia treatment, and having broad application prospects and important clinical significance.

[0020] The anti-leukemia composition provided by the present invention can further broaden its therapeutic scope and improve the therapeutic effect by combining PDC-type PROTAC with other active ingredients with anti-leukemia effects; the addition of pharmaceutically acceptable carriers or excipients also helps to improve the stability and bioavailability of the composition. The anti-leukemia composition has multiple active ingredients that can exert a synergistic effect and improve the therapeutic effect of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of the method for synthesizing enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptides targeting human leukemia cells of the present invention; Figure 2 is the MALDI-TOF-MS spectrum of HIF-IMA; Figure 3 is the MALDI-TOF-MS spectrum of Cyclo-C9C-R-HIF-IMA; Figure 4 is the MALDI-TOF-MS spectrum of Cyclo-C9C-R-GFLG-HIF-IMA; Figure 5 This is a diagram of the release process of the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide targeting human leukemia cells of the present invention (under the condition of the presence of enzyme); Figure 6 This is a result diagram showing the effect of the PDC-type PROTAC modified with the cell-penetrating peptide targeting human leukemia cells of the present invention on THP1 cell viability; Figure 7 This is a result diagram showing the inhibitory activity of the PDC-type PROTAC modified with the human leukemia cell-targeting transmembrane peptide of the present invention on K562 cell proliferation; Figure 8 This is a result diagram showing the inhibitory activity of the PDC-type PROTAC modified with the human leukemia cell-targeting transmembrane peptide of the present invention on KU812 cell proliferation; Fig. 9 It is a result diagram of the degradation effect of the PDC-type PROTAC modified with the human leukemia cell-targeted penetrating peptide of the present invention on the target protein in K562 cells; wherein A is a result diagram of the degradation effect of HIF-IMA on the target protein in K562 cells, C is a result diagram of the degradation effect of Cyclo-C9C-R-HIF-IMA on the target protein in K562 cells, E is a result diagram of the degradation effect of Cyclo-C9C-R-GFLG-HIF-IMA on the target protein in K562 cells, and B, D, and F are semi-quantitative results of the target protein expression levels in A, C, and E; Fig.10 The results of the degradation effect of the PDC-type PROTAC modified with a human leukemia cell-targeted penetrating peptide on the target protein in KU812 cells are shown in Figure 1; wherein, A is the result of the degradation effect of HIF-IMA on the target protein in KU812 cells, C is the result of the degradation effect of Cyclo-C9C-R-HIF-IMA on the target protein in KU812 cells, E is the result of the degradation effect of Cyclo-C9C-R-GFLG-HIF-IMA on the target protein in KU812 cells, and B, D and F are the semi-quantitative results of the target protein expression levels in A, C and E, respectively; Fig.11The effect of the enzyme-sensitive PDC-type PROTAC modified with the cell-penetrating peptide targeting human leukemia cells of the present invention on the apoptosis of K562 cells; wherein A is the effect of HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on the apoptosis of K562 cells, and B is the quantitative result of apoptotic cells in A; Fig.12 The effect of the enzyme-sensitive PDC-type PROTAC modified with the cell-penetrating peptide targeting human leukemia cells of the present invention on the apoptosis of KU812 cells; wherein A is the effect of HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on the apoptosis of KU812 cells; B is the quantitative result of apoptotic cells in A; Fig.13 The effect of PDC-type PROTAC on the cell cycle of K562 cells; A is the effect of HIF-IMA on the cell cycle of K562 cells; B is the quantitative result of A; Fig.14 The effect of the enzyme-sensitive PDC-type PROTAC modified with the cell-penetrating peptide targeting human leukemia cells of the present invention 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; Fig.15 The effect of PDC-type PROTAC on the KU812 cell cycle; A is the effect of HIF-IMA on the KU812 cell cycle; B is the quantitative result of A; Fig.16 The figure shows the effect of the enzyme-sensitive PDC-type PROTAC modified with the cell-penetrating peptide targeting human leukemia cells of the present invention on the cell cycle of KU812 cells; wherein A is the effect of Cyclo-C9C-R-GFLG-HIF-IMA on the cell cycle of KU812 cells, and B is the quantitative result of A. DETAILED DESCRIPTION

[0022] The present invention provides a human leukemia cell targeting penetrating peptide modified PDC-type PROTAC, which is obtained by modifying the PDC-type PROTAC (the sequence is shown in SEQ ID NO.1 in Table 1) with a human leukemia cell targeting penetrating peptide and an enzyme sensitive sequence. The sequence of the human leukemia cell targeting penetrating peptide modified PDC-type PROTAC is shown in SEQ ID NO.2 to SEQ ID NO.3 in Table 1.

[0023] The PROTAC is obtained by connecting a hypoxia-inducible factor-1α protein fragment to a drug molecule through dodecanedioic acid, and the sequence of the hypoxia-inducible factor-1α protein fragment is LAP(OH)YI (as shown in SEQ ID NO.5).

[0024] Among them, the human leukemia cell targeting transmembrane peptide is Cyclo-C9C-R (as shown in SEQ ID NO.4), and the enzyme sensitive sequence is the cathepsin B sensitive sequence GFLG.

[0025] Table 1: Amino acid sequence listing

[0026] Note: In the amino acid sequence, "L" stands for "Leucine", "A" stands for "Alanine", "P(OH)" stands for "Hydroxy-L-proline", "Y" stands for "Tyrosine", "I" stands for "Isoleucine", "IMA" stands for "Imatinib", "C(....)" stands for cyclization, "K" stands for "Lysine", "R" stands for "Arginine", "F" stands for "Phenylalanine", "W" stands for "Tryptophan", "R" stands for "Arginine", "S" stands for "Serine", "E" stands for "Glutamic acid", and "G" stands for "Glycine".

[0027] 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.

[0028] 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, unless otherwise specified, "%" represents weight percentage.

[0029] 1. Experimental Materials 1. Experimental drugs and reagents: Rink Amide-MBHA resin (B&K), Fmoc-protected amino acids, piperidine (Sinopharm Group), N,N-dimethylformamide (DMF, Komyo Chemical Reagent), DCM (Komyo Chemical Reagent), HATU (Biode Pharmaceuticals), HOBt (Biode Pharmaceuticals), DIPEA (Biode Pharmaceuticals), (benzotriazol-1-yloxy)tripyrrolidino hexafluorophosphate (PyBop, Biode Pharmaceuticals), triethylamine (TFA, Aladdin), TIS (Aladdin), 1,3-dimethoxybenzene (Aladdin), DODT (Aladdin), chromatography acetonitrile (Aladdin), PBS buffer (CORNING), RPMI 1640 (CORNING), penicillin-streptomycin solution (double antibody) (MCE), fetal bovine serum (NEWZERUM), Trypsin (CORNING), DMSO (MP Biomedicals), and MTT (Solarbio).

[0030] 2. Experimental cells: K562 (human chronic myeloid leukemia cells), KU812 (human peripheral blood basophilic leukemia cells) and THP1 (human leukemia monocytic cell line).

[0031] 2. Synthesis of enzyme-sensitive PDC-type PROTACs modified with cell-penetrating peptides targeting human leukemia cells like Figure 1 As shown, an enzyme-sensitive PDC-type PROTAC modified with a transmembrane peptide targeting human leukemia cells was synthesized using a solid-phase synthesis method based on FMOC amino acids.

[0032] 1. Conjugation of N-desmethylimatinib with dodecanedioic acid N - Coupling of demethylimatinib with dodecanedioic 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) Dodecanedioic 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; N-Demethylimatinib (0.47 g, 0.98 mmol) in anhydrous DMF, monitor the system pH ≈ 8, and react at room temperature for 4 hours; after the reaction is completed, water is added, and the mixture is extracted with ethyl acetate (3×), and the combined organic phase is washed with brine, dried over Na2SO4, and evaporated under reduced pressure to obtain the corresponding crude product; column chromatography is used to separate the obtained off-white solid (0.55 g, 82% yield), mass spectrum (ESI, m / z): 692.35 [M + H] + , 690.30 [M - H] - ,get N - Coupling product of demethylimatinib and dodecanedioic acid: 12-(4-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)carbamoyl)benzyl)piperazin-1-yl)-12-oxododecanoic acid.

[0033] 2. Synthesis of Cyclo-C9C-R-GFLG-HIF-IMA (1) Resin preparation and swelling Rink Amide-MBHA resin (50.0 mg, 0.32 mmol / g, 0.016 mmol) was placed in a reaction vessel (centrifugal column), and 5 mL of a mixed solvent of DMF / DCM (1:1) was added to allow the resin to fully swell for 45 min, and then the filtrate was discarded by vacuum filtration; (2) Deprotection reaction Add 20% piperidine / DMF mixed solution and perform FMOC protection group removal reaction for 10 minutes. Filter the filtrate under reduced pressure. Repeat the above operation twice to completely remove the Fmoc protecting group at the N-terminus of Rink Amide-MBHA resin. After filtering, wash the resin thoroughly with DCM / DMF (1:1) mixed solution to remove residual piperidine and DMF. (3) Amino acid connection Weigh 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 mixed solution, add the mixed solution to the centrifugal column, react for 45 minutes, so that Fmoc-Glu-Oall is successfully connected to the resin, filter and thoroughly wash the resin with DCM / DMF (1:1) to remove unreacted amino acids and reagents, and connect amino acids in sequence according to the sequences shown in SEQ ID NO.1 to SEQ ID NO.3 in Table 1, and repeat the above connection and washing steps; (4) Cyclization reaction For the peptides that needed cyclization, after the deprotection reaction was completed, 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 reactions were carried out twice, with the time being 3 h and 8 h, respectively, to ensure the complete cyclization reaction. (5) Coupling product connection After completing the synthesis of the peptide part, N -The coupling product of demethylimatinib and dodecanedioic 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 coupling agent DIC (12.4 μL, 0.08 mmol) were added to the centrifuge column and reacted on a rotating mixer overnight to successfully connect the coupling product to the polypeptide chain to form a complete Cyclo-C9C-R-GFLG-HIF-IMA molecule; (6) Product cleavage and purification After the synthesis is completed, the cleavage reagent (V TFA : V TIS : V 1,3-二甲氧基苯 : V DODT : V H2O = 90:2.5:2.5:2.5:2.5) to cleave the product from the resin, the cleavage agent can destroy the connection bond between the resin and the polypeptide chain and release the product; DCM is added to the centrifuge column to thoroughly wash the product and collect the liquid, dissolve the product and remove the residual cleavage agent; evaporate the solvent, add icy ether to the centrifuge tube, the product is precipitated, the product is centrifuged at 8000 rpm for 20 minutes, and then the supernatant is discarded; add icy ether to the centrifuge tube again, gently wash the product, centrifuge at 8000 rpm for 5 minutes, discard the supernatant to obtain the crude peptide; store the crude peptide in a -80°C refrigerator for subsequent purification.

[0034] (7) Purification and characterization See attached Figures 2~4The 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 a 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, and the purity of the purified peptide was detected at a wavelength of 254 nm to ensure that the purity was >90.0%.

[0035] Through the analysis of MALDI-TOF-MS spectra, the correct synthesis, purity and structural characteristics of HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA molecules were confirmed, providing a reliable basis for subsequent biological research and applications.

[0036] 3. Release detection of enzyme-sensitive PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells High performance liquid chromatography was used to detect the release of PDC-PROTAC in the prepared human leukemia cell-targeted transmembrane peptide-modified enzyme-sensitive PDC-type PROTAC. The specific method is as follows: The reaction solution of Cyclo-C9C-R-GFLG-HIF-IMA was 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, 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 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours to prepare HPLC samples (Shimadzu LC-2030C 3D Plus). The changes in the peak height and peak area of ​​Cyclo-C9C-R-GFLG-HIF-IMA / HIF-IMA were observed respectively. Chromatographic conditions: Shimadzu C18 column (C18-120 / 5μm, 10.0×250 mm), mobile phase: 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℃, detection volume: 50 μL, detection wavelength: 254 nm.

[0037] The release properties of PDC-PROTAC in the enzyme-sensitive PDC-type PROTAC modified with a transmembrane peptide targeting human leukemia cells Figure 5 shown.

[0038] from Figure 5It can be seen that Cyclo-C9C-R-GFLG-HIF-IMA (sequence as shown in SEQ ID NO.3 in Table 1) achieves rapid release of HIF-IMA under the catalysis of cathepsin B, with a release of 46.22% within 1 hour, more than 90% of HIF-IMA can be released in 4 hours, and 98.74% of HIF-IMA can be released within 6 hours. These results show that the connection of human leukemia cell-targeted transmembrane peptides to PDC-PROTACs through enzyme-sensitive linkers can achieve full release of PDC-PROTACs.

[0039] Detection of cytotoxicity of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells The MTT method was used to determine the cytotoxicity of PDC-type PROTAC modified with human leukemia cell-targeted transmembrane peptides on tumor cells and control cells. The specific method is as follows: 1. Cell inoculation: Dilute K562 cells, KU812 cells and THP1 cells in the exponential growth phase to 10 4 Cell solutions at the order of 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 cultured at 37°C, 5% CO2 for 12 h.

[0040] 2. Drug administration: Add 20 μL of HIF-IMA, Cyclo-C9C-R-HIF-IMA or Cyclo-C9C-R-GFLG-HIF-IMA of different concentrations to each well to obtain a final concentration of 3.0×10 -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 replicate wells for each concentration and 6 replicate wells for the negative control, and continue to culture at 37°C, 5% CO2 for 48 h.

[0041] 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, 5% CO2 for 4 h, carefully remove the supernatant, add 150 μL of DMSO to each well, shake for 10 min, measure the ultraviolet absorbance (OD value) of each well at 490 nm with an enzyme-linked immunosorbent assay, and then calculate the cell inhibition rate; The calculation formula of cell inhibition rate is: Inhibition rate % = (average OD value of control wells - average OD value of drug group) / average OD value of control wells × 100%; 4. Experimental results: Figure 6 As can be seen, the cytotoxicity analysis showed that 3.0×10 -1 In the concentration range of μmol / L to 100.0 μmol / L, the compounds HIF-IMA, Cyclo-C9C-R-HIF-IMA or Cyclo-C9C-R-GFLG-HIF-IMA had little effect on the cell viability of K562 cells, KU812 cells and THP1 cells, and the cell survival rates were all above 80%, proving that this type of human leukemia cell-targeted transmembrane peptide-modified PDC-type PROTAC has a certain safety at the cellular level.

[0042] 5. Detection of tumor cell proliferation inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells K562 cells and KU812 cells in good growth state and logarithmic growth phase were centrifuged at 1200rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium and inoculated into 96-well cell culture plates at a seeding volume of 5000 cells per well, with a seeding volume of 180 μL. Three wells were set for each sample, and blank wells and negative controls were set. After inoculation, the culture plate containing the cells was placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA or Cyclo-C9C-R-GFLG-HIF-IMA was selected and configured into a certain concentration gradient solution, and administered to K562 cells and KU812 cells at a dose of 20 μL per well. After 48 hours of administration, MTT solution was added, and then placed in an incubator at 37°C and 5% CO2 for further culture for 4 hours. The cell inhibition rate of the cells was calculated after measuring the absorbance.

[0043] The calculation formula of cell inhibition rate is: Inhibition rate % = (average OD value of control wells - average OD value of drug group) / average OD value of control wells × 100%; The tumor cell proliferation inhibitory activity of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells Figure 7 and Figure 8 shown.

[0044] from Figure 7 and Figure 8It can be seen that the proliferation inhibitory activity of Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on K562 cells and KU812 cells is better than that of HIF-IMA, and their proliferation inhibitory activity increases with the increase of the administration concentration. At low concentrations (concentrations of 0.3 μmol / L and 1.0 μmol / L), the proliferation inhibitory activity of Cyclo-C9C-R-HIF-IMA on K562 cells is significantly higher than that of HIF-IMA, and the proliferation inhibitory activity of HIF-IMA can be increased by 5.67 times and 7.01 times, respectively. These results show that the introduction of human leukemia cell-targeted transmembrane peptides can significantly enhance the proliferation inhibitory activity of HIF-IMA on K562 cells. In addition, the introduction of enzyme-sensitive Linker (GFLG) also promotes the anti-proliferative activity of HIF-IMA, and is applicable at all concentrations. These results further demonstrate the rationality of the design of PDC-type PROTAC modified with human leukemia cell-targeted transmembrane peptides.

[0045] VI. Detection of protein degradation activity of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells K562 cells and KU812 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 . Three wells were set for each sample, and blank wells and negative controls were set. After inoculation, the culture plate containing the cells was placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA, Cyclo-C9C-R-GFLG-HIF-IMA and imatinib were selected and configured 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.

[0046] The results of protein degradation activity of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells are as follows Figures 9 and 10 shown.

[0047] from Fig. 9 and Fig.10It can be seen that HIF-IMA has poor degradation activity on the target protein in K562 cells and KU812 cells. When its concentration is 30 μmol / L, it can only degrade 22.55% of BCR-ABL protein in K562 cells, and has no significant effect on the phosphorylation of downstream STAT5. In KU812 cells, even when the concentration of HIF-IMA reaches 50 μmol / L, it can only degrade 20.01% of BCR-ABL protein. However, after modification with human leukemia cell-targeted penetrating peptides, the degradation activity of Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on the target protein in K562 cells and KU812 cells was significantly improved. In K562 cells, when the concentration was 30 μmol / L, Cyclo-C9C-R-HIF-IMA had a slightly higher degradation activity on BCR-ABL protein than HIF-IMA, and could degrade 28.66% of BCR-ABL protein. When the concentration of Cyclo-C9C-R-HIF-IMA was 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. Moreover, the degradation activity of Cyclo-C9C-R-HIF-IMA on target protein in KU812 cells was also better than that of HIF-IMA, which could not only enhance the degradation of BCR-ABL protein by HIF-IMA, but also enhance the inhibition of downstream STAT5 phosphorylation.

[0048] In addition, the use of enzyme-sensitive linkers can significantly improve the degradation effect of HIF-IMA on target proteins, and it can work at low concentrations. Specifically, in K562 cells, when the concentration of Cyclo-C9C-R-GFLG-HIF-IMA is 0.3μmol / L, compared with Cyclo-C9C-R-HIF-IMA, the degradation rate of BCR-ABL protein can be increased by 1.79 times, and the inhibition of downstream STAT5 phosphorylation can also be promoted to a certain extent. In KU812 cells, Cyclo-C9C-R-GFLG-HIF-IMA can significantly improve the inhibition of STAT5 phosphorylation, a downstream protein of BCR-ABL. The above results further prove that the introduction of enzyme-sensitive linkers can enhance the degradation activity of HIF-IMA on target proteins, confirming the applicability of this strategy to hematological tumor cells. The results of the investigation on the protein degradation mechanism of HIF-IMA showed that the proteasome inhibitor MG-132 can block the protein degradation induced by Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA, among which the blocking effect on the protein degradation induced by Cyclo-C9C-R-GFLG-HIF-IMA is more obvious, indicating that it acts through the proteasome pathway.

[0049] VII. Detection of apoptosis of human leukemia cells by PDC-type PROTAC modified with cell-penetrating peptides K562 cells and KU812 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 100 cells / well. Three wells were set for each sample, and blank wells and negative controls were set. After inoculation, the culture plates containing cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. HIF-IMA, Cyclo-C9C-R-HIF-IMA, and Cyclo-C9C-R-GFLG-HIF-IMA were selected and configured 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, Cyto FLEX).

[0050] The results of apoptosis detection of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells are as follows Fig.11 and Fig.12 shown.

[0051] from Fig.11 and Fig.12 It can be seen that compared with HIF-IMA, the ability of Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA to induce apoptosis of K562 cells was significantly improved, and they had good effects when administered at low and high concentrations. When the administration concentration was 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. When the administration concentration was 30 μmol / L, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA had a stronger effect on the induction of apoptosis of K562 cells, which was 10.53 times and 12.09 times that of HIF-IMA, respectively. In addition, Cyclo-C9C-R-GFLG-HIF-IMA has a stronger effect on inducing apoptosis of K562 cells than Cyclo-C9C-R-HIF-IMA. When the dosage concentration is 10 μmol / L, the apoptosis induction effect of Cyclo-C9C-R-GFLG-HIF-IMA is 1.85 times that of Cyclo-C9C-R-HIF-IMA. The apoptosis induction effect of HIF-IMA, Cyclo-C9C-R-HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA on KU812 cells is weak, but Cyclo-C9C-R-GFLG-HIF-IMA still has the best ability to promote apoptosis of KU812 cells. The above results show that after modification with human leukemia cell-targeted transmembrane peptides and enzymatic linkers, the pro-apoptotic effect of HIF-IMA on tumor cells is significantly improved.

[0052] 8. Cell cycle detection of human leukemia cells with PDC-type PROTAC modified with cell-penetrating peptides K562 cells and KU812 cells in the optimal growth condition and logarithmic growth phase were cultured at 10 μg / well. 6The 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 and negative controls were set. After inoculation, the culture plates containing cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. HIF-IMA and Cyclo-C9C-R-GFLG-HIF-IMA were selected and configured as 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, samples were analyzed on a flow cytometer and data were analyzed using the NovoExpress software package.

[0053] The results of cell cycle detection of PDC-type PROTAC modified with cell-penetrating peptide targeting human leukemia cells are as follows Figures 13 to 16 shown.

[0054] from Figures 13 to 16 It can be seen that compared with HIF-IMA, the application of Cyclo-C9C-R-GFLG-HIF-IMA blocks K562 cells in the G1 / M phase. When the concentration is 30 μmol / L, Cyclo-C9C-R-GFLG-HIF-IMA can block 69.87% of K562 cells in the G1 / M phase, which is higher than the effect of HIF-IMA at the same concentration (59.64%). For KU812 cells, Cyclo-C9C-R-GFLG-HIF-IMA blocks them in the S phase. When the concentration is 30 μmol / L, Cyclo-C9C-R-GFLG-HIF-IMA can block 64.15% of KU812 cells in the S phase, which is higher than the effect of HIF-IMA at the same concentration (55.05%).

[0055] Based on the above, it can be seen that the enzyme-sensitive PDC-type PROTAC modified with human leukemia cell-targeted transmembrane peptide can be used to prepare anti-leukemia drugs (targeted protein degradation and transmembrane penetration), has certain leukemia cell targeting and transmembrane penetration capabilities, and has little effect on the cell viability of K562 cells, KU812 cells and THP1 cells, has proliferation inhibitory activity on K562 cells and KU812 cells, can degrade target proteins in K562 cells and KU812 cells, and can induce apoptosis of K562 cells and KU812 cells, and affect their cell cycle. The modification of human leukemia cell-targeted transmembrane peptides and enzyme-sensitive sequences can expand the structural diversity of PROTAC. At the same time, activity tests show that the introduction of human leukemia cell-targeted transmembrane peptides and enzyme-sensitive sequences plays an important role in improving the protein degradation effect of PDC-PROTAC, which can increase the application scenarios of PDC-PROTAC and can serve as another important field for PROTAC development.

[0056] 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 human leukemia cell-targeted cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC, characterized in that: The enzyme-sensitive PDC-type PROTAC is obtained by modifying PROTAC with a human leukemia cell-targeted transmembrane peptide; the target protein ligand in the PROTAC is an anti-leukemia drug; The human leukemia cell targeting membrane-penetrating peptide sequence is shown in SEQ ID NO.4; The sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.

2.

2. A human leukemia cell-targeted cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 1, characterized in that: The enzyme-sensitive PDC-type PROTAC also includes an enzyme-sensitive sequence, which is obtained by modifying PROTAC with a human leukemia cell-targeted transmembrane peptide and an enzyme-sensitive sequence, and the amino acid sequence of the enzyme-sensitive sequence is a cathepsin B-sensitive sequence.

3. A human leukemia cell-targeted cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to claim 2, characterized in that: The sequence of the enzyme-sensitive PDC-type PROTAC is shown in SEQ ID NO.

3.

4. A human leukemia cell-targeting cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to any one of claims 1 to 3, characterized in that: When the human leukemia cell-targeted penetrating peptide is used alone for modification, the human leukemia cell-targeted penetrating peptide is linked to PROTAC via a covalent bond; When the human leukemia cell targeting transmembrane peptide and the enzyme-sensitive sequence are used for co-modification, the human leukemia cell targeting transmembrane peptide, the enzyme-sensitive sequence and PROTAC are linked by covalent bonds.

5. A human leukemia cell-targeting cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to any one of claims 1 to 4, characterized in that: The PROTAC is obtained by connecting a hypoxia-inducible factor-1α protein fragment to a drug molecule through dodecanedioic acid, and the sequence of the hypoxia-inducible factor-1α protein fragment is shown in SEQ ID NO.

5.

6. A method for preparing a human leukemia cell-targeting cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to any one of claims 1 to 5, 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.

7. Use of the human leukemia cell-targeting cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to any one of claims 1 to 5 in the preparation of anti-leukemia drugs.

8. The use according to claim 7, characterized in that: The anti-leukemia drug is a drug targeting human chronic myeloid leukemia cells or human peripheral blood basophilic leukemia cells.

9. The use according to claim 7, characterized in that: The concentration of the enzyme-sensitive PDC-type PROTAC modified with a cell-penetrating peptide targeting human leukemia cells was 3.0×10 -1 ~100.0 μmol / L.

10. An anti-leukemia composition, characterized in that: The anti-leukemia composition comprises other active ingredients having anti-leukemia effects, the human leukemia cell-targeting cell-penetrating peptide-modified enzyme-sensitive PDC-type PROTAC according to any one of claims 1 to 5, and one or more pharmaceutically acceptable carriers or excipients.

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