Synthesis and application of membrane splitting peptide Citropin and drug conjugate

Abstract

The invention belongs to the field of biological medicines, and provides a preparation method and application of a stapled peptide and micromolecular antitumor drug covalent conjugate with high stability and strong antitumor activity. Traditional micromolecular antitumor drugs such as camptothecin, chlorambucil and the like have the problems of low water solubility, low bioavailability, poor stability, drug resistance and the like. The camptothecin or chlorambucil is covalently coupled with the stapled peptide through AEA, so that the water solubility of the small molecule medicine is remarkably improved, the anti-tumor onset time is remarkably shortened, and the long-lasting tumor cell proliferation inhibition capability is shown. Compared with linear peptide, the stability of the stapled peptide is remarkably improved, and a polypeptide drug coupling strategy based on the stapled peptide solves the problem of poor stability of polypeptide in a coupled drug on one hand, and provides a new thought for further development of classical anti-tumor drugs on the other hand.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of polypeptide preparation and biological medicine, and particularly relates to a preparation method of a conjugate based on membrane cleavage stapled peptide and small molecule antitumor drug and application thereof. BACKGROUND

[0002] Cancer, as the core challenge in the field of global public health in the 21st century, has posed a serious threat to the economic development of human society due to the continuous rise in its incidence. According to epidemiological statistics, there were 170 million new cases of malignant tumors (excluding non-melanoma skin cancer) worldwide in 2018, of which about 9.8 million patients needed to receive chemotherapy. It is predicted that by 2040, the annual number of new tumor cases will increase to 26 million, and the size of the population of patients with indications for chemotherapy is expected to expand to 15 million. Although targeted therapy and immunotherapy technologies are rapidly developing, chemotherapy is still the first-line treatment for patients with advanced cancer and is widely used as a key adjuvant in combination therapy. However, due to inherent defects, traditional chemotherapy drugs have limited efficacy, and it is urgent to break through the bottleneck through technological innovation.

[0003] For example, camptothecin (CPT) exerts antitumor activity by inhibiting DNA topoisomerase I (TOP I), but the physicochemical properties of its rigid pentacyclic skeleton result in three defects: the α-hydroxy lactone ring is easily hydrolyzed and inactivated under physiological conditions, has extremely low water solubility (limited bioavailability), and has non-selective toxicity to rapidly proliferating normal tissues. Similarly, the alkylating agent representative drug chlorambucil (CLB) can cross-link with DNA to induce tumor cell apoptosis, but is limited by poor water solubility, non-selective cytotoxicity-induced myelosuppression, and short half-life due to insufficient membrane permeability. The above common defects highlight the need for optimization of traditional chemotherapy drugs.

[0004] Peptide-drug conjugates (PDCs) as a new strategy, through the optimization of drug solubility, membrane permeability, and synthetic economy by polypeptide carriers, have become a very promising alternative. Cationic anticancer peptides are particularly suitable for PDCs design due to their ability to effectively bind to anion-rich cancer cell membranes. The Citropin1.1 derivative A4K14-Citropin1.1 (805) modified by alanine (A4) and lysine (K14) has enhanced α-helix stability and membrane cleavage activity, but its linear structure is easily degraded by proteases and has insufficient conformational stability, limiting its direct application.

[0005] In view of the polypeptide stability problem, the stapled peptide technology fixes the alpha-helix conformation through chemical modification, which is an effective means to improve the drugability of polypeptides. Therefore, previous studies have optimized the structure of A4K14-Citropin 1.1 linear peptide through stapled peptide strategy to develop a lead compound for new cancer treatment drugs. Among them, A4K14-Citropin 1.1-Sp4 is a full-hydrocarbon chain stapled peptide derivative of A4K14-Citropin 1.1, which has significantly improved helicity and exhibits stronger protease stability and anti-tumor activity. However, existing studies have not fully explored the potential of stapled peptides as PDCs carriers, especially in coupling classic chemotherapeutic drugs such as CPT and CLB to synergistically enhance anti-tumor effects, which still has a technical gap. SUMMARY

[0006] The present application provides a preparation method and application of a class of stapled peptides covalently coupled with small molecule anti-tumor drugs with high stability and strong anti-tumor activity. Traditional small molecule anti-tumor drugs such as CPT and chlorambucil (CLB) have problems such as low water solubility, low bioavailability, poor stability, and drug resistance. Compared with linear peptides, stapled peptide A4K14-Citropin 1.1-Sp4 has significantly improved stability and exhibits strong anti-tumor activity. The present application modifies stapled peptide A4K14-Citropin 1.1-Sp4 with 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) and further covalently couples it with small molecule chemotherapeutic drugs to improve the anti-tumor ability of small molecule drugs, wherein the small molecule drugs are preferably CPT and CLB.

[0007] After covalently coupling CPT or CLB with the hybrid peptide, the water solubility is significantly improved, showing a significant 72h sustained tumor inhibition effect, and the activity is significantly improved compared with free drugs, and the in vivo anti-tumor activity is significantly better than that of linear peptides. It can be seen that the stapled peptide-drug conjugate of the present application provides a new approach for the development of classic anti-tumor drugs, and has good practical application value.

[0008] Specifically, the present application is realized by the technical solutions as follows:

[0009] In a first aspect, the present application provides a polypeptide as a conjugate of a membrane-lytic stapled peptide and an N-terminal conjugation linker AEEA. The polypeptide preferably adopts A4K14-Citropin 1.1-Sp4 modified by stapling strategy and AEEA-modified polypeptide (polypeptide 849), which exhibits higher serum stability and cell proliferation inhibition activity than the original peptide. The amino acid sequence and chemical structure formula are as follows:

[0010]

[0011] In a second aspect, the present application provides polypeptide conjugates, preferably CPT and CLB. CPT and CLB are conjugated to polypeptide 849 to obtain conjugates 851 and 852. The amino acid sequence abbreviations and chemical structures are as follows:

[0012]

[0013] In a third aspect, the present application provides a method for preparing polypeptide conjugates, comprising the following steps:

[0014] (1) Stapled peptide synthesis method, the present application provides a normal temperature method 9-fluorenylmethyloxycarbonyl (Fmoc) - solid phase polypeptide synthesis technology and olefin metathesis reaction combined synthesis stapled peptide method, preferably RinkAmide AM resin. When synthesizing polypeptide sequence main chain containing special amino acids, HCTU / DIEA condensation system and HATU / HOAt / DIEA condensation system are preferred.

[0015] (2) AEEA stapled peptide synthesis, preferably Fmoc-based solid phase polypeptide synthesis method is used, RinkAmide AM resin is preferred, and HCTU / DIEA condensation system and HATU / HOAt / DIEA condensation system are preferred for synthesis.

[0016] (3) Method for coupling with small molecule drugs, the present application provides a Fmoc-based solid phase polypeptide synthesis method, and HCTU / DIEA condensation reaction is preferably used for covalent coupling of small molecule drugs. Small molecule drugs are preferably CPT or CLB, which are coupled with 849. CPT is modified by succinic acid, and is coupled with amino groups on the target polypeptide through amide bond. CLB is coupled with amino groups on the target polypeptide through carboxyl groups thereon.

[0017] In a fourth aspect, the present application provides the use of the polypeptide conjugates in tumor treatment.

[0018] The results obtained through in vitro cytotoxicity experiments and time-inhibition rate determination experiments show that the N-terminal AEEA-coupled all-carbon chain stapled peptide not only exhibits strong in vitro anti-tumor activity, but also has anti-tumor durability. Through serum stability experiments, it is found that the AEEA stapled peptide has better proteolytic stability than the original peptide A4K14-Citropin 1.1. Especially importantly, hemolysis experiments show that the N-terminal connection of AEEA effectively improves the biocompatibility of the all-carbon chain stapled peptide at the working concentration. The present application provides a carrier polypeptide with good tumor killing effect, good stability and high safety for the development of PDC drugs.

[0019] Secondly, the application finds that the hybrid peptide 851 / 852 covalently coupled with CPT or CLB shows a significant 72h sustained tumor inhibition effect, and the activity is significantly improved compared with 849 and free drugs (CPT / CLB). Through the water solubility determination experiment, it is found that compared with free drugs, the water solubility of the conjugate 851 / 852 is significantly improved, which provides a key experimental basis for improving the bioavailability of CPT / CLB. Finally, the application finds that the conjugate 851 covalently coupled shows a significant ability to inhibit tumor growth in mice, which provides a new idea for the further development of classic antitumor drugs

[0020] The beneficial technical effects of the above technical solution are as follows:

[0021] The application not only provides an innovative solution to overcome the bottlenecks of poor solubility and slow effect of traditional chemotherapy drugs, but also clarifies the synergistic mechanism of stapled peptide modification and introduction of hydrophilic linking groups through multi-dimensional structure-function analysis, thereby laying a practical application value for developing efficient and stable polypeptide-drug conjugate systems. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The structural formula, primary amino acid sequence, reversed-phase high performance liquid chromatogram and ESI-MS mass spectrum of the stapled peptide 849 of the application are shown.

[0023] Figure 2 The structural formula, primary amino acid sequence, reversed-phase high performance liquid chromatogram and ESI-MS mass spectrum of the hybrid peptide 851 of the application are shown.

[0024] Figure 3 The structural formula, primary amino acid sequence, reversed-phase high performance liquid chromatogram and ESI-MS mass spectrum of the hybrid peptide 852 of the application are shown.

[0025] Figure 4 The time-inhibition rate curves of A4K14-Citropin 1.1, A4K14-Citropin 1.1-Sp4 and 849 of the application are shown.

[0026] Figure 5 The serum stability determination graphs of A4K14-Citropin 1.1, A4K14-Citropin 1.1-Sp4 and 849 of the application are shown.

[0027] Figure 6 The hemolysis rate determination graphs of A4K14-Citropin 1.1, A4K14-Citropin 1.1-Sp4 and 849 of the application are shown.

[0028] Figure 7 The water solubility determination graphs of 851\CPT(A) and 852\CLB(B) of the application are shown.

[0029] Figure 8 Inhibition of body weight (A) and tumor growth (B) in tumor-bearing mice by the prototype peptide 805 of the present application, and its derivative peptides 849, 851 and low-dose 851. DETAILED DESCRIPTION

[0030] To better illustrate the present application, the following examples are given:

[0031] Example 1 Solid-phase synthesis of polypeptides 849, 851, 852

[0032] First, the appropriate amount of Rink-Amide AM resin was accurately weighed according to the reaction amount, and the following operations were performed in sequence: after standard washing with N,N-dimethylformamide (DMF) and dichloromethane (DCM), the resin was soaked and swelled in analytical pure DMF for 1-2 h to complete the initial activation treatment. Then, 3-4 mL of DMF / DCM mixed solvent (v:v = 4:1) was used to activate the resin again, and the resin was placed in a constant temperature shaker (28°C, 120 rpm) for shaking for 30-60 min to ensure that the resin was fully activated. After completing the activation procedure, the Kaiser color reaction was first performed to monitor whether the Fmoc group was completely removed, and then the condensation reaction was strictly carried out according to the amino acid sequence.

[0033] Method for amino acid condensation reaction of polypeptide 849: The amino acid and condensation reagent were dissolved in analytical pure DMF and ultrasonically mixed well and then transferred to a synthesis tube. The condensation reaction was carried out step by step under constant temperature conditions of 28°C: condensation was carried out twice, and the reaction time of the second condensation was appropriately prolonged to ensure complete reaction. For general amino acid condensation, the condensation reaction ratio of Fmoc-amino acid:HCTU:DIEA was 3 eq:2.8 eq:6 eq, and the reaction time was 40 min and 60 min; for S configuration pentenyl alanine (Fmoc-(S)-2-(4-pentenyl)Ala-OH, S5) and AEEA, the HATU / HOAt / DIEA condensation reaction system was selected, wherein the reaction ratio of S5 was Fmoc-amino acid:HATU:HOAt:DIEA = 1.5 eq:1.3 eq:1.5 eq:3 eq, and AEEA was adjusted to 2 eq:1.8 eq:2 eq:4 eq, and the corresponding condensation times were set to 60 / 80 min and 60 / 120 min, respectively. After completing the synthesis of the polypeptide chain, 1 eq of the product was dissolved in 2.5 eq of the first generation Grubbs' catalyst (Grubbs' first-generation catalyst) in anhydrous dichloroethane (DCE), transferred to a synthesis tube, and subjected to intramolecular cyclization reaction at 28°C for 4 h.

[0034] The amino acid condensation reaction method of polypeptide 851 / 852: the above cyclized polypeptide chains are then coupled with CPT and CLB, respectively. The coupling reaction of CPT and the main chain is carried out in a material ratio of CPT-COOH:HATU:HOAt:DIEA = 2 eq:1.8 eq:2 eq:4 eq, and needs to be carried out twice, with a duration of 70 min and 90 min, respectively. The coupling of CLB and the main chain is realized by a HCTU / DIEA condensation reaction system, with a reaction material ratio of CLB:HCTU:DIEA = 3 eq:2.8 eq:6 eq, also needing two reactions, with a time of 70 min and 90 min, respectively.

[0035] The ice bath peptide cleavage reagent [trifluoroacetic acid (TFA): phenyl hydroxide (PhOH): water: triisopropylsilane (TIPS) = 85:5:5:2 (v:v:v:v)] is added to the cyclization product system, and constant temperature shaking reaction is carried out at 28°C in the dark for 1.5-2 h. After the reaction is terminated, the mixture is transferred to a three-necked flask through a synthesis tube, and then the resin is rinsed with a small amount of TFA and the eluate is collected. Excess TFA is removed by bubbling high-purity nitrogen gas to reduce the volume of the system to 2-3 mL. At this time, pre-cooled anhydrous ether is slowly added to the concentrated solution, and the target peptide forms a precipitate. The precipitate is obtained by centrifugation (3000 rpm, 3 min), and the supernatant is discarded to obtain the crude peptide product. The above precipitation-centrifugation operation is repeated three times, and finally the crude product is transferred to a fume hood for drying treatment.

[0036] The crude peptide solution is freeze-dried in a freeze-drier, and the freeze-dried powder is dissolved and then separated and purified by semi-preparative RP-HPLC. After a round of freeze-drying, the target pure peptide is obtained.

[0037] The crude peptide product is dissolved in a 0.1% TFA-containing acetonitrile / water mixed solvent system, completely dissolved after ultrasonic treatment, and then analyzed and identified by analytical reversed-phase high performance liquid chromatography (RP-HPLC) combined with ESI-MS. Then the crude product is pre-frozen at -80°C, and transferred to a freeze-drier to obtain a loose powder. The freeze-dried powder is re-dissolved, separated and purified by semi-preparative RP-HPLC, and the target peak fraction is collected and freeze-dried again to obtain a high-purity target polypeptide.

[0038] Figure 1 、 Figure 2 and Figure 3The amino acid sequences and structural formulas of polypeptides 849, 851 and 852, and the RP-HPLC chromatograms and corresponding ESI-MS mass spectra of the purified polypeptides are shown in the figures below. Time-inhibition rate curve, serum stability and hemolytic activity of 849

[0039] Time-inhibition rate curve determination experiment

[0040] A20 cell suspension in the logarithmic growth phase was diluted with 1640 culture medium to a density of 1 x 10 4 cells / well, 100 μL of the cell culture medium mixture was added to each well, and the 96-well plate was incubated overnight in a constant-temperature cell incubator at 37°C and a CO2concentration of 5%. Next, the working concentrations of the polypeptides were determined according to the IC 50 values of each polypeptide, 20 μM for A4K14-Citropin 1.1, and 10 μM for A4K14-Citropin 1.1-Sp4 and 849. The polypeptide working solutions diluted with serum-free 1640 culture medium were added to the 96-well plate, and three replicate wells were set up for each polypeptide sample. A blank control group was also set up, and the plate was incubated in the incubator for 4 h, 12 h, 24 h, 36 h and 48 h. After treatment for different lengths of time, the CCK8 method was used for detection.

[0041] Different structural modifications have a significant impact on the anti-tumor persistence of polypeptides. The dashed line represents a stapled peptide with long-term anti-tumor activity, while the solid line represents a linear peptide with short-term activity. Linear peptide A4K14-Citropin 1.1 showed a significant downward trend from 12 h to 48 h, with an inhibition rate of less than 11% at 24 h. In sharp contrast, the stapled peptide modified by a full hydrocarbon chain (A4K14-Citropin 1.1-Sp4, 849) exhibited excellent time-dependent properties, with a tumor inhibition rate of more than 80% from 12 h to 48 h Figure 4 . Time-inhibition rate curve analysis showed that carbon chain cyclization modification can improve the anti-cancer persistence of A4K14-Citropin 1.1, which may be related to improved hydrolytic protein stability.

[0042] Serum stability determination experiment

[0043] Take 200 μL of 1 mM polypeptide stock solution and add to PBS buffer containing 5% human serum, shake gently 3 times and mix well to prepare polypeptide solution with final concentration of 200 μM. Incubate the mixture at 37°C, at selected time points (0, 2, 4, 16, 24, 36, 48 and 72 h), take 50 μL of reaction mixture, quench the proteolysis reaction with mixed solution (50 μL of 0.08%-0.1% TFA in acetonitrile, 250 μL of 0.1% TFA in water) and store at -20°C until analysis. Finally, all samples were centrifuged at low temperature and high speed (4°C, 12000 rpm, 20 min) and the supernatant was treated with 0.22 μm microfiltration membrane and analyzed by RP-HPLC.

[0044] The experimental results are shown in Table 1. Figure 5 As shown in Table 1, the structural integrity of the three polypeptides showed time-dependent decrease after incubation in 5% human serum for 72 h. At 24 h, the remaining amount of linear peptide A4K14-Citropin 1.1 was only 48.6%, while the remaining amount of the structurally modified A4K14-Citropin 1.1-Sp4 and 849 was 71.6% and 75.8%, respectively. At 72 h, the remaining amount of A4K14-Citropin 1.1, A4K14-Citropin 1.1-Sp4 and 849 was 28.8%, 63.7% and 69.5%, respectively. The half-life of linear peptide A4K14-Citropin 1.1 in 5% human serum was about 24 h, while the half-life of stapled peptides A4K14-Citropin 1.1-Sp4 and 849 was more than 72 h under the same conditions. The results of serum stability assay showed that the all-carbon hydrogen chain stapled peptides A4K14-Citropin 1.1-Sp4 and 849 showed good stability compared to linear peptide A4K14-Citropin 1.1, and their remaining amount of polypeptide at 72 h was about twice that of the original peptide A4K14-Citropin 1.1.

[0045] Hemolysis assay experiment

[0046] The polypeptide mother liquor with a concentration of 10 mM was serially diluted in 1x PBS to prepare polypeptide stock solutions with concentrations of 200, 60, 20, 6, 2, 0.6, and 0.2 μM, respectively. 200 μL of each solution was aliquoted into sterile 1.5 mL EP tubes (at this time, the concentration was twice the final required concentration). Subsequently, 200 μL of a 4% red blood cell suspension was added to the EP tubes containing an equal volume of the polypeptide stock solution, and the final concentration of the peptide was 0.1-100 μM. The mixture was incubated at 37°C for 4 h. 0.1% Triton X-100 and PBS (1x) were used as positive and negative controls, respectively. After incubation, the supernatant was obtained by centrifugation (1500 rpm, 5 min). Finally, the collected supernatant was transferred to a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader. All samples were tested in triplicate to ensure the reliability and accuracy of the data.

[0047] The results are shown in Table 1. Figure 6 As shown in Table 1, the hemolysis rates of the three polypeptides were all less than 15% at low concentrations (<1 μM). As the concentration increased to the range of 3-100 μM, all three peptides showed dose-dependent hemolysis, but the polypeptides with different structural modifications showed significant differences. Analysis of the experimental results showed that when the polypeptide concentration was higher than 3 μM, the hemolysis rate of A4K14-Citropin 1.1-Sp4 increased to 35.9%, while the hemolysis rate of A4K14-Citropin 1.1 was less than 5%. This was because the cyclization modification of the all-hydrocarbon chain increased the hydrophobicity of the polypeptide, causing severe hemolysis.

[0048] Notably, the hemolysis rate of 849 modified by the AEEA linker was lower than that of A4K14-Citropin 1.1-Sp4 in the high concentration range of 3-100 μM. In particular, when the polypeptide concentration was 10 μM, the hemolysis rate of A4K14-Citropin 1.1-Sp4 increased to 69.8%, while the hemolysis rate of 849 (30.4%) was only 43.5% of the former. Studies have shown that the introduction of a hydrophilic AEEA linker at the N-terminus of the all-carbon hydrogen chain stapled peptide can effectively improve the water solubility of the stapled peptide molecule and significantly reduce the hemolyticity, which provides an important theoretical basis for optimizing the biocompatibility of polypeptide compounds.

[0049] Based on the above research results, the stapled peptide 849 modified by the AEEA hydrophilic linker not only retains the anti-tumor durability and proteolytic stability of the stapled peptide, but also significantly reduces the high hemolyticity of the all-carbon hydrogen chain stapled peptide due to the introduction of the AEEA linker. Therefore, 849 was selected as a carrier for small molecule chemotherapeutic drugs, and was coupled with CPT and CLB to construct hybrid peptides to improve the anti-tumor activity of small molecule chemotherapeutic drugs

[0050] Example 3 Evaluation of the ability to inhibit cell proliferation

[0051] The MTT colorimetric method was used to evaluate the inhibitory effect of the synthetic peptide on HepG2 and Hela tumor cells. The above cells were seeded in 96-well plates with a cell density of 10 cells per well. 4 Then, incubate in a cell culture incubator overnight. 50 μL of basal culture medium with peptide concentrations of 1, 3, 10, 30, 100, and 300 μM was added to each well and incubated for 24 / 48 / 72 hours, respectively. After the incubation, 15 μL of MTT (5 mg / mL) was added to each well and incubated in a cell culture incubator for 4 hours. The supernatant was removed and 150 μL of DMSO was added to each well. After incubation for 1 hour, the absorbance was measured at 492 nm using a microplate reader. IBM SPSS Statistics 27 software was used to calculate the half-maximal inhibitory concentration (IC) values ​​obtained from three independent parallel experiments. 50 ) and visualized the dose-effect curves using GraphPad Prism 9.0 software. All IC 50 Values ​​are expressed as mean ± SEM.

[0052] As shown in Table 1, the single CPT-modified hybrid peptide 851 significantly shortened the duration of CPT's action. At 72 hours, 851 exhibited 4-15-fold greater anticancer activity against HepG2 and HeLa tumor cells than 849, with sustained tumor killing. This indicates that conjugation of 849 with CPT not only shortened CPT's onset time but also significantly enhanced its anticancer activity.

[0053] 852 was synthesized by covalently linking CLB with 849. 852 is more soluble than free CLB (IC 50 The anticancer efficacy was significantly improved compared to that of 849 (>100μM), with slightly higher anticancer activity at 24 hours than that of 849, and a stable tumor inhibitory effect was maintained for 72 hours. These results demonstrate that conjugating 849 to small molecule anticancer drugs is an effective and universal strategy for enhancing the antitumor activity of small molecule drugs, providing new insights for the further development of classic anticancer drugs.

[0054] Table 1 Incubation of the test substances with tumor cells for 24 h a and 72h b After IC 50 Value (μM)

[0055]

[0056] Example 4 Water Solubility

[0057] First, the full wavelength spectrum scanning (200-800 nm) of the target polypeptide with known concentration was performed using a UV-2600 type ultraviolet visible spectrophotometer, and the detection wavelength of each substance was determined by locating the characteristic absorption peak. To determine the saturation solubility, the absorbance value of the saturated solution was measured at the respective detection wavelength. The actual concentration was calculated according to the standard curve equation, and multiplied by the dilution factor to obtain the final solubility value. All experiments were set up in triplicate to ensure data reliability. This analysis method combines characteristic wavelength selection and standard curve quantification to achieve accurate characterization of the solubility characteristics of compounds.

[0058] The experimental data showed that the average water solubility of CPT and CLB was 3.8 μM and 45 μM, respectively, while the average water solubility of 851 and 852 reached 9.1 mM and 12.6 mM, respectively, which was about 2300 times and 280 times higher than that of free CPT and CLB, respectively. Figure 7 In summary, 851 and 852 significantly improved the solubility of small molecule drugs CPT and CLB, providing valuable reference for the modification and development of traditional small molecule drugs.

[0059] Example 5 Anti-tumor experiment in mice

[0060] A 6-7 week old male Balb / c mouse was used to construct a tumor-bearing animal model, and about 1×10 7 4T1-LUC cells were inoculated in the left armpit of each mouse. After tumor formation, the mice were randomly assigned to five treatment groups (n = 6 per group) and given the following intratumoral injection treatments: normal saline, 805 (0.5 mg per mouse), 849 (0.5 mg per mouse), 851 (0.5 mg per mouse), and 851 low-dose group (0.25 mg per mouse). The dosing regimen was to inject every 48 h for two weeks. During the entire experiment, the body weight changes of the mice were monitored and recorded every 48 h. At the end of the experiment, all animals were euthanized, and the tumor tissue was removed and weighed.

[0061] As shown in Figure 8 The 851 low-dose group showed certain in vivo anti-tumor activity, with tumor weight significantly less than the 805 and 849 treatment groups. Overall, 851 effectively inhibited tumor growth in mice, and the high-dose group was significantly better than the low-dose group. During the entire experiment, the body weight of the mice did not decrease significantly, indicating that the mice were well tolerated to the treatment dose of polypeptide Figure 8 A). The in vivo experiment in mice showed that the covalent conjugate 851 exhibited stronger anti-tumor growth effect than the original staple peptide, providing a candidate compound for the development of high-efficiency anti-tumor drugs.

Claims

1. Preparation of a polypeptide drug conjugate, characterized in that: The polypeptide drug conjugate comprises a covalent conjugate of a membrane-cleaving stapled peptide A4K14-Citropin 1.1-Sp4 and a small molecule tumor chemotherapy drug.

2. The polypeptide drug conjugate according to claim 1, characterized in that The polypeptide is coupled to the drug via a linker; preferably, the linker is 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

3. The polypeptide drug conjugate according to claim 1, characterized in that: The drug is a small molecule tumor chemotherapy drug; preferably, the small molecule tumor chemotherapy drug is camptothecin or chlorambucil; The stapled peptide-camptothecin conjugate has the chemical structural formula: The chemical structural formula of the stapled peptide-chlorambucil conjugate is:

4. The method for preparing the polypeptide drug conjugate according to claim 1 and claim 3, characterized in that: The method for connecting the polypeptide and the linking group, and the linking group and the drug molecule is a solid-phase polypeptide synthesis method; the stapling peptide is a method of combining solid-phase polypeptide synthesis technology and olefin metathesis reaction.

5. Use of the polypeptide drug conjugate according to claim 1 in a drug for preventing and / or treating tumor-related diseases, characterized in that: The tumor disease is one or more of liver cancer, cervical cancer or malignant lymphoma, or a solid tumor resistant to traditional chemotherapy drugs.

6. A pharmaceutical preparation, characterized in that It comprises the conjugate according to claim 1 and pharmaceutically acceptable excipients; Preferably, the excipients include diluents, fillers, disintegrants, surfactants, suspending agents, binders, lubricants, colorants and flavorings.

Images