Stapling peptide, p53-MDM2 / MDMX dual inhibitor and application of p53-MDM2 / MDMX dual inhibitor

Abstract

The invention discloses a binding peptide, a p53-MDM2 / MDMX dual inhibitor and application of the p53-MDM2 / MDMX dual inhibitor, and belongs to the technical field of biological medicines. The amino acid sequence of the binding peptide is LTFKcHYWKcQLTSAA, and amino groups of two lysine residues in the sequence are crosslinked. The invention provides a promising strategy for recovering p53 functions and preventing tumor growth by developing an MDM2 / MDMX dual inhibitor, including a lysine stapled peptide inhibitor designed based on key residues of p53 helix, to activate a p53 pathway. Compared with a traditional binding strategy, the binding peptide provided by the invention has obvious advantages in the aspects of serum stability, cellular uptake ability, water solubility, toxicity and the like in tumor treatment. Meanwhile, the binding peptide is proved to have remarkable anti-tumor activity and high safety, and the limitation of the binding peptide in clinical application in the prior art is solved, so that the binding peptide has a wide application prospect in the field of cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicines, and in particular relates to a stapled peptide, a p53-MDM2 / MDMX dual inhibitor and uses thereof. Background Art

[0002] In the field of drug development, α-helical peptides with stapled structures have attracted much attention due to their potential to target protein-protein interactions (PPIs) in a variety of diseases (Zhang, YJ Med. Chem. 2023, 66 (1), 95-106; Cheng, J. RSC Med. Chem. 2023, 14 (12), 2496-2508). Since Blackwell and Grubbs first proposed the concept of stapled peptides in 1998, and Verdine and his colleagues further promoted the development of this field through pioneering research (Verdine, Science (New York, NY) 2004, 305 (5689), 1466-1470), stapled peptides have been widely studied as probe compounds for evaluating PPI targets (Modi, R, J. Med. Chem. 2023, 66 (9), 6184-6192). However, stapled peptides face a series of challenges in the process of translation into clinical research. These challenges mainly include optimizing their physicochemical and pharmacological properties, such as achieving high affinity target binding, improving cell permeability, improving solubility, reducing serum protein binding, and adjusting in vivo pharmacokinetic properties to ensure their stability and effectiveness in vivo (Lee, MF, Pharm. Res. 2023, 40 (3), 617-632). To overcome these obstacles, medicinal chemists have adopted a variety of strategies, one of which is to expand the chemical space of fixed peptides by diversifying the "stapled" structure. Currently, a variety of chemical tools have been developed for the synthesis of stapled peptides, including but not limited to cysteine-based reactions (Fairlie, DP, Biopolymers 2016, 106(6), 843-852), alkyne-azide click reactions (Lau, YH, Nat. Protoc. 2015, 10(4), 585-594), ring-closing olefin metathesis (Walensky, LD, J. Med. Chem. 2014, 57(15), 6275-6288), lactamization, etc. (Taylor, JW, Biopolymers 2002, 66(1), 49-75). Nevertheless, the development of new stapled peptides to meet clinical needs remains an urgent task.

[0003] In the field of biomedicine, the development of inhibitors targeting specific protein-protein interactions is an important direction for cancer treatment research. In particular, p53 protein and its interaction with MDM2 and MDMX play a key role in regulating tumor growth (Joerger, A.C. Annu. Rev. Biochem. 2016, 85, 375-404; Wade, M. Nat. Rev. Cancer 2013, 13 (2), 83-96). Historically, researchers have derived initial peptide sequences from the p53 N-terminal α-helical domain and developed a variety of stapled peptides to enhance their stability and biological activity. For example, Verdine and colleagues identified a stapled peptide SAH-p53-8, which is a fully carbon cross-linked peptide formed by a ring-closing metathesis reaction. Although it showed cytotoxicity against SJSA-1 osteosarcoma cancer cells under serum-free conditions, it was not effective enough under standard cell culture conditions (Bernal, FJ Am. Chem. Soc. 2007, 129 (9), 2456-2457). In addition, although the subsequent development of stapled peptides has improved affinity, the water solubility problem limits its further clinical research.

[0004] In order to overcome the shortcomings of the existing technology, Chen and his colleagues discovered a dual-target inhibitor pDI, which has high affinity for both MDM2 and MDMX (Hu, B. Cancer Res. 2007, 67 (18), 8810-8817; Phan, JJ Biol. Chem. 2010, 285 (3), 2174-2183.). Based on pDI, the stapled α-helical peptide ATSP-3900 was further developed, which has significantly enhanced binding affinity for MDM2 and MDMX. ATSP-7041 was optimized by peptide sequence mutation, which exhibited high affinity and strong cell activity under standard cell culture conditions (Chang, YS Proc. Natl. Acad. Sci. USA 2013, 110 (36), E3445-E3454.). However, the water solubility problem of ATSP-7041 limits its clinical application.

[0005] Although the above-mentioned stapling strategies have made certain progress in biomedical applications such as antibacterial and anti-tumor, different stapling methods still differ in terms of serum stability, cell uptake ability, water solubility and toxicity (Xiang Li, Chem. Rev. 2020, 120, 18, 10079–10144), which limits its effectiveness in clinical applications. Although the existing stapling strategies have improved the stability and biological activity of peptides to a certain extent, there are still some limitations. For example, some stapled peptides are not stable enough in serum, resulting in a short half-life in the body; or their cellular uptake ability is limited, which affects the bioavailability of the drug; in addition, some stapled peptides have poor water solubility, which may affect their preparation and administration; and some stapled peptides have certain toxicity, which limits the safety of their clinical application. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a stapled peptide, a p53-MDM2 / MDMX dual inhibitor and uses thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] A stapled peptide, the amino acid sequence of which is: LTFK c HYWK c QLTSAA (SEQ ID NO.1, K c Indicates a lysine cross-linking site), and the side chain amino groups of two lysine residues in the sequence are cross-linked, and its specific structure is as follows:

[0009]

[0010] Furthermore, the crosslinking group used in the crosslinking is 1,3-dimethylenephenyl.

[0011] Furthermore, the N-terminus of the stapled peptide is acetylated and the C-terminus is amidated.

[0012] A preparation targeting MDM2 and MDMX proteins, comprising the above-mentioned stapled peptide.

[0013] Furthermore, the agent can bind to MDM2 and / or MDMX proteins in cells.

[0014] A dual inhibitor of p53-MDM2 / MDMX protein interaction, comprising the above-mentioned stapled peptide, or preparation.

[0015] Furthermore, the dual inhibitor can bind to MDM2 and / or MDMX proteins in cells, activate p53 protein, and increase the expression level and / or activity of p53 protein.

[0016] Use of the above-mentioned stapled peptide, preparation, or dual inhibitor in the preparation of inhibitors or drugs for treating tumors.

[0017] Further, the tumor is a tumor caused by excessive accumulation of MDM2 and / or MDMX in p53-MDM2 / MDMX.

[0018] Furthermore, the tumor is breast cancer, colorectal cancer or non-small cell lung cancer.

[0019] A pharmaceutical composition comprises the above-mentioned stapled peptide SPDI-48-T1 targeting p53-MDM2 / MDMX protein-protein interaction, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention provides a promising strategy for restoring p53 function and preventing tumor growth by developing MDM2 / MDMX dual inhibitors, including lysine-stapling peptide inhibitors designed based on key residues of the p53 helix, to activate the p53 pathway. Compared with the traditional stapling strategy, the novel lysine-stapling peptide inhibitor of p53-MDM2 / MDMX interaction of the present invention has obvious advantages in serum stability, cell uptake ability, water solubility and toxicity in tumor treatment. The establishment of in vitro anti-proliferation experiments and ectopic xenograft tumor treatment models further confirmed that it has significant anti-tumor activity and high safety. It solves the limitations of stapled peptides in clinical applications in the prior art, and thus has broad application prospects in the field of cancer treatment.

[0022] 2. The present invention provides a novel lysine-stapled peptide derivative of a linear pDI peptide, which exhibits stronger serum stability, cell permeability, higher water solubility, significant in vitro and in vivo anti-tumor activity and safety compared to the linear pDI peptide. Preliminary tests have found that the novel lysine-stapled peptide inhibitor SPDI-48-T1 disclosed above has good in vitro and in vivo anti-tumor activity and has potential prospects for drug development.

[0023] 3. The present invention obtains a novel lysine-stapled peptide inhibitor SPDI-48-T1 having a 1,3-dimethylene cross-linked structure by a novel and efficient preparation method. The synthesis method is simple and the preparation cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A roadmap for the preparation of the novel lysine-stapled peptide inhibitor SPDI-48-T1;

[0025] Figure 2 This is a graph showing the stability results of the novel lysine-stapled peptide inhibitor SPDI-48-T1 in 50% human serum;

[0026] Figure 3 The K of the novel lysine-stapled peptide inhibitor SPDI-48-T1 and its target protein MDM2 / MDMX d Value determination.

[0027] Figure 4 This is the result of laser confocal microscopy experiment on cell membrane penetration of the novel lysine-stapled peptide inhibitor SPDI-48-T1;

[0028] Figure 5 This is a graph showing the mouse tumor volume and relative tumor volume growth in the SPDI-48-T1 mouse xenograft tumor model, a novel lysine-stapled peptide inhibitor;

[0029] Figure 6 This is a graph showing the tumor weight of mice after treatment with the novel lysine-stapled peptide inhibitor SPDI-48-T1 in an ectopic xenograft mouse tumor model;

[0030] Figure 7 This is the solid anatomical diagram of mouse tumors after treatment with the new lysine-stapled peptide inhibitor SPDI-48-T1. DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0032] Example 1 Preparation of the novel lysine-stapled peptide inhibitor SPDI-48-T1

[0033] 1. Preparation of linear peptide I: In a solid phase synthesis tube filled with 330 mg Rink AM resin (loading amount 0.67 mmol / g), the amino acid sequence LTFKHYWKQLTSAA was coupled to the resin step by step from C to N terminus using solid phase peptide synthesis (SPPS) technology.

[0034] 2. Preparation of Compound II:

[0035] 1) 0.22 mmol of resin was loaded into a 12 mL solid phase synthesis tube.

[0036] 2) Add 7 mL of tetrahydrofuran (THF) to the synthesis tube.

[0037] 3) Add 869 μL of a 40% aqueous solution of tetrabutylammonium hydroxide (TBAH, 1.33 mmol, 6 eq).

[0038] 4) Place the synthesis tube on a shaker and shake at 270 rpm for 10 minutes.

[0039] 5) 352 mg of 1,3-bis(bromomethyl)benzene (1.33 mmol, 6 eq) were added.

[0040] 6) After sealing, continue shaking for 6-8 hours.

[0041] 7) The resin was washed with 3×10 mL of N,N-dimethylformamide (DMF), 3×10 mL of methanol (MeOH), and 3×10 mL of dichloromethane (DCM) in sequence.

[0042] 8) Analyze the resin sample using liquid chromatography-mass spectrometry (LC-MS) to confirm complete coupling and good purity

[0043] 3. Preparation of compound III:

[0044] 1) Add 7 mL of N,N-dimethylformamide (DMF) to a 12 mL solid phase synthesis tube containing compound II.

[0045] 2) 340 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2.22 mmol, 10 eq) and 82 μL of β-mercaptoethanol (1.11 mmol, 5 eq) were added in sequence.

[0046] 3) Place the solid phase tube on a shaker and shake for 8 hours.

[0047] 4) After the reaction was completed, the resin was washed with 3×10 mL N,N-dimethylformamide (DMF), 3×10 mL methanol (MeOH), and 3×10 mL dichloromethane (DCM) in sequence.

[0048] 5) Analyze the resin sample again using LC-MS to confirm complete coupling and good purity.

[0049] 4. Preparation of compound IV:

[0050] 1) In a 12 mL solid phase tube containing compound IV, 10 mL of a cleavage reagent mixture was added, wherein the mixture consisted of trifluoroacetic acid (TFA), triethylsilane (TES), and water (H2O) in a volume ratio of 95:2.5:2.5.

[0051] 2) Place the solid phase tube on a shaker and shake at room temperature for 8-10 hours.

[0052] 3) After the reaction is completed, transfer the cutting solution to a 50 mL centrifuge tube and use an air pump or nitrogen to blow the cutting solution dry to remove the solvent and recover the crude target polypeptide (see Figure 1 ).

[0053] 5. Separation and purification of compound IV:

[0054] 1) Add 20 mL of pre-cooled ether to a 50 mL centrifuge tube containing compound IV and perform ultrasonic treatment to promote dissolution.

[0055] 2) Centrifuge at 3200 rpm for 10 minutes to precipitate the crude polypeptide.

[0056] 3) After centrifugation, carefully pour off the supernatant to avoid disturbing the precipitate, and use nitrogen to blow dry the ether to further purify the target compound.

[0057] 4) Add 12 mL of pure water and 3 mL of acetonitrile or methanol to the precipitate and perform ultrasonic treatment again to dissolve the target compound.

[0058] 5) Prefreeze the solution in a -80°C refrigerator overnight to prepare for freeze-drying.

[0059] 6) The pre-frozen sample was quickly transferred to a freeze dryer and freeze-dried at -99°C and 33 mF for 3-4 days to obtain a crude product.

[0060] 7) Separate the crude product by preparative liquid chromatography (RP-HPLC) to obtain a high-purity target compound. Analyze the purity by high-performance liquid chromatography (HPLC) to ensure that the purity of the compound meets the experimental requirements.

[0061] 8) Liquid chromatography-mass spectrometry (LC-MS) and quadrupole time-of-flight liquid-mass spectrometry system (high-resolution Q-TOF MASS) were used for analysis to confirm the molecular weight and structure of the compound.

[0062] 9) Collect samples that meet the purity standards and perform vacuum concentration to remove the solvent. Pre-freeze the concentrated samples in a -80°C refrigerator overnight to prepare for freeze-drying.

[0063] 10) The pre-frozen sample was transferred to a freeze dryer and freeze-dried at -99°C and 33 mT for 3-4 days to obtain the target compound.

[0064] Example 2: Evaluation of water solubility of compound SPDI-48-T1

[0065] The water solubility of a compound is one of the important indicators for evaluating its bioavailability, efficacy and stability. A systematic study of the water solubility of a specific compound is of great significance for optimizing drug formulations and improving therapeutic effects. Therefore, this example aims to determine and verify the water solubility of the new lysine-stapled peptide inhibitor SPDI-48-T1 under specific conditions through precise experimental design. The specific process is as follows

[0066] 1. Experimental Materials

[0067] Compound sample: novel lysine-stapled peptide inhibitor SPDI-48-T1, with a purity of 98%.

[0068] Solvent: Ultrapure water

[0069] 2. Experimental steps

[0070] At room temperature (25°C), use an electronic balance to accurately weigh the compound sample SPDI-48-T1, weigh 5.0 mg, 2.5 mg, and 0.5 mg of the sample and place them in a 0.5 mL EP tube. Add 50 μL of ultrapure water to each of the three samples to make the sample concentrations reach 100 mg / mL, 50 mg / mL, and 10 mg / mL, respectively. Shake the EP tube thoroughly to ensure that the compound is fully dissolved. It was observed that the solutions of the three samples were all clear and transparent, with no residual solids, indicating that the compound was completely dissolved at the corresponding concentration.

[0071] 3. Experimental results

[0072] Through this experiment, at concentrations of 100 mg / mL, 20 mg / mL and 10 mg / mL, the compound SPDI-48-T1 can be completely dissolved in ultrapure water to form a clear and transparent solution. It can be inferred that the solubility of the compound in ultrapure water at room temperature is greater than 100 mg / mL, indicating that it can exhibit good solubility in ultrapure water at room temperature.

[0073] Example 3: Serum stability test

[0074] The peptides were incubated at 37°C at a concentration of 1.28 mg / mL in phosphate buffered saline (PBS) containing 50% human serum. Aliquots of 50 μL were taken at regular intervals at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours and added to 100 μL of a mixed solution of H2O / MeCN (1:3) containing 12% trichloroacetic acid, and then cooled at 4°C for 30 minutes. Subsequently, all samples were centrifuged (12,000 rpm, 10 minutes) to remove precipitated proteins. The supernatant was analyzed by high-performance liquid chromatography (HPLC), monitoring the UV absorption at 220 nm and recording the integrated peak area. A standard curve of integrated area and peptide concentration was established by treating the peptide with PBS containing 50% human serum and then immediately adding a mixed solution of H2O / MeCN (1:3) containing 12% trichloroacetic acid and analyzing it by HPLC. Using the standard curve, the percentage of intact peptide was calculated by determining the integrated area. All experiments were repeated three times, and the average value was reported. The results are shown in Figure 2 .

[0075] like Figure 2 As shown, the novel lysine-stapled peptide inhibitor SPDI-48-T1 prepared by the present invention was co-incubated with 50% human serum, and the measured half-life was about 12 hours.

[0076] Example 4: Fluorescence polarization assay to determine the K of the novel lysine-stapled peptide inhibitor SPDI-48-T1 and the target protein MDM2 / MDMX d value

[0077] Fluorescence polarization analysis was used to evaluate the binding affinity (K) of the novel lysine-stapled peptide inhibitor SPDI-48-T1 for MDM2 and MDMX. d The specific process is as follows:

[0078] MDM2 and MDMX were diluted with PBS to different concentration gradients ranging from 7.8nM to 4μM. The FAM-labeled novel lysine-stapled peptide inhibitor was dissolved in PBS with a starting concentration of 1mM. It was then diluted to 4nM with PBS. In a 384-well black well plate, 50μL of different concentrations of MDM2 or MDMX and 50μL of the FAM-labeled novel lysine-stapled peptide inhibitor SPDI-48-T1 were added to each well. The mixture was incubated at 37°C for 4 hours, and the fluorescence polarization value was recorded by a microplate reader. The following formula is used to calculate K by nonlinear fitting d value:

[0079]

[0080] Where y represents the measured fluorescence polarization value; X represents the concentration of MDM2 or MDMX protein; C represents the final concentration of FAM-labeled peptide; F maxrepresents the maximum fluorescence polarization value; F min Represents the minimum fluorescence polarization value.

[0081] like Figure 3 As shown in Figure 2, the binding affinity (K) of the novel lysine-stapled peptide inhibitor SPDI-48-T1 prepared by the present invention to MDM2 and MDMX d The values ​​of (a) and (b) were 396 nM and 456 nM, respectively, which showed high binding affinity to the target proteins MDM2 and MDMX.

[0082] Example 5: Evaluation of cell membrane uptake capacity

[0083] Hoechst and DiD fluorescent dyes were used to evaluate the cell membrane permeability of peptides. The specific process is as follows:

[0084] HCT116 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 2×10 5 The cells were seeded in a 96-well plate at a density of , and then cultured overnight in a 5% CO2, 37°C constant temperature incubator. DMEM was then removed, and the cells were treated with 200 μL of 40 μM fluorescein (FAM)-labeled polypeptide (FAM-SPDI-48-T1) in DMEM for 4 hours. After treatment, the culture medium was aspirated and the cells were washed three times with phosphate buffered saline (PBS). Then, the cells were fixed with 4% paraformaldehyde (200 μL) for 10 minutes and then washed three times with phosphate buffered saline (PBS). Next, the cells were stained with Hoechst 33342 at a concentration of 5 μg / mL in a volume of 200 μL, incubated for 15 minutes in the dark, and then washed three times with phosphate buffered saline (PBS). The cells were further stained with DiD at a concentration of 10 μM in a volume of 200 μL, incubated for 30 minutes in the dark, and then washed three times with phosphate buffered saline (PBS). Anti-fluorescence quenching agent was added to the cover, and the image was observed using a laser confocal scanning microscope. The results are shown in Figure 2. Figure 4 .

[0085] like Figure 4 As shown, the novel lysine-stapled peptide inhibitor SPDI-48-T1 prepared by the present invention can efficiently penetrate the cell membrane and enter the cell.

[0086] Example 6: Antitumor Activity Test

[0087] In order to investigate the anti-tumor activity of SPDI-48-T1 prepared by the present invention, the inhibition of tumor growth by SPDI-48-T1 was preliminarily evaluated by an in vitro anti-tumor cell proliferation test. The in vitro test model of tumor cells treated with SPDI-48-T1 was used, and the MTT method was used to determine the proliferation activity of tumor cells and compare and screen the anti-tumor activity of SPDI-48-T1. (The reagents and materials involved can be obtained through public channels and belong to the common knowledge in the field.) The test operation steps include:

[0088] (1) Cell culture

[0089] MCF-7 (human breast cancer cells), HCT116 (human colon cancer cells), A549 (human non-small cell lung cancer cells), cells were cultured in Dulbecco's modified Eagle's medium (DMEM). ZR-75-1 (human breast adenocarcinoma cells) cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640). All culture media were supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution (Penicillin-Streptomycin), and cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0090] (2) Drug preparation

[0091] All compounds were prepared immediately before use, with the highest concentration being 160 μM. The compounds were prepared with sterile double distilled water, completely dissolved, filtered through a 0.22 μM sterile syringe filter for sterilization, and stored at -20°C. When administering, dilute with sterile PBS in multiples according to the required drug concentration.

[0092] (3) MTT method

[0093] Take the required cells in the logarithmic growth phase, adjust to the appropriate concentration and inoculate them in a 96-well culture plate, 100 μL per well (about 3000-10000 cells), and incubate at 37°C, 5% CO2 for 24 hours. According to the required final drug concentration, dilute the highest concentration drug with sterile PBS, and then add the prepared drugs to each experimental well of the culture plate in turn, 100 μL per well, so that the final concentration is 5 μM, and 5 replicates are set for each concentration. The negative control group is an equal volume of PBS solvent control added to the same volume of culture medium. After administration, the 96-well culture plate is incubated at 37°C, 5% CO2 for 72 hours.

[0094] MTT method was used to determine cell proliferation activity: 100 μL of MTT (500 μg / mL) was added to each well of a 96-well cell culture plate and mixed well. The plate was incubated at 37°C and 5% CO2 for 4 h. The culture medium was then carefully aspirated to reveal the blue-purple precipitate at the bottom of the plate. 100 μL of DMSO was added to each well and gently shaken to dissolve the precipitate. The OD value of each well was measured using an ELISA reader. 490 The values ​​were calculated and the inhibition rate was calculated. The statistical differences were statistically analyzed and the results are shown in Table 1.

[0095] Table 1 SPDI-48-T1 antitumor activity test results

[0096]

[0097] As shown in Table 1, the novel lysine-stapled peptide inhibitor SPDI-48-T1 prepared by the present invention exhibits significant anti-tumor activity and has potential pharmaceutical uses.

[0098] Example 6: Testing the in vivo efficacy and toxicity of SPDI-48-T1 in an ectopic xenograft tumor model in immunodeficient mice

[0099] The in vivo antitumor activity of SPDI-48-T1 was evaluated by establishing an ectopic xenograft tumor model in immunodeficient mice. In the experiment, immunodeficient mice were randomly assigned to different treatment groups, including a saline control group and two dose groups of SPDI-48-T1 (5 mg / kg and 10 mg / kg), to investigate the in vivo therapeutic effect of SPDI-48-T1 at different doses. The experimental operation steps include:

[0100] 1) Model establishment: An ectopic xenograft tumor model was established in immunodeficient mice to simulate the growth environment of human tumors.

[0101] 2) Drug administration: The mice were treated with SPDI-48-T1 at doses of 5 mg / kg and 10 mg / kg body weight, respectively, and a normal saline control group was set up.

[0102] 3) Tumor growth monitoring: Tumor volume was measured and recorded regularly to evaluate the effect of SPDI-48-T1 on tumor growth.

[0103] 4) Tumor tissue collection: During the experiment, the changes in the mouse tumor volume were recorded; after the experiment, the mouse tumor tissue was removed, the tumor volume and weight were recorded, and the tumor tissue was photographed and recorded.

[0104] 5) Histological evaluation: The heart, liver, spleen, lung, and kidney of mice were sectioned and then stained with H&E to evaluate the potential toxicity of SPDI-48-T1.

[0105] 6) Animal Ethics and Compliance: During the animal experiments of this invention, we strictly abide by the internationally recognized animal ethics principles and relevant laws and regulations. All experimental animals used are from legal and certified suppliers to ensure the quality and welfare of the animals. The experimental animals are raised in SPF (Specific Pathogen Free) animal breeding rooms and cared for by professionals to ensure the ethics of the experiments and the health of the animals.

[0106] like Figure 5 As shown in the figure, the tumor volume measurement results during the experimental period (D1-D15) showed that the volume of HCT116 transplanted tumors in the vehicle control group continued to grow, and the average tumor volume increased from 118.14±22.94mm on the day of grouping (D1) to 3 The growth to the end point of the experiment (D15) was 1574.34±301.71mm 3 The relative tumor volume (RTV) increased from 1.00 to 13.33±2.74, indicating that the human colorectal cancer HCT116 nude mouse subcutaneous transplant tumor model was successfully established in this experimental system, which is suitable for the needs of tumor proliferation inhibition experimental research.

[0107] The test product SPDI-48-T1 had a certain inhibitory effect on the proliferation of HCT116 transplanted tumors under the set treatment regimen (5 mg / kg and 10 mg / kg, tail vein administration, once every two days, 8 doses). Compared with the solvent control group during the same period, the tumor volume growth of animals in the SPDI-48-T1 high-dose group showed a significant inhibition state, and the average tumor volume increased from 118.47±11.26mm on the day of grouping to 118.47±11.26mm on the day of grouping. 3 Growth to the end point of the test: 912.14±189.14mm 3 The relative tumor volume increased from 1.00 to 7.70±1.77, which was significantly different from the tumor growth process of the animals in the vehicle control group during the same period (P<0.05); the tumor volume growth of the low-dose SPDI48-T1 group showed an inhibitory trend, but there was no significant statistical difference compared with the tumor growth process of the animals in the vehicle control group during the same period.

[0108] The tumor-bearing animals were euthanized at the end of the experiment, and the tumor tissue was collected and weighed. Figure 6 and Figure 7 As shown, the low-dose group of the test article had no obvious inhibitory trend on the proliferation of HCT116 transplanted tumors; the high-dose group of the test article had a significant inhibitory effect on the proliferation of HCT-116 transplanted tumors. Compared with the solvent control group (1.401±0.278g) in the same period, the tumor weight of animals in the high-dose group (0.981±0.129g) showed a significant decrease, and the tumor proliferation inhibition rate (TGI) was 30.02%, and the difference was statistically significant (P<0.05).

[0109] Analysis of the H&E staining results of heart, liver, spleen, lung and kidney tissue sections showed that both the high-dose group and the low-dose group were toxic to the main organs of mice, and there were no obvious pathological changes in the tissues, which proved that the new lysine-stapled peptide inhibitor SPDI-48-T1 prepared by the present invention has efficient in vivo anti-tumor activity and high in vivo safety; it provides a compound with important application prospects in the field of drug development.

[0110] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A stapled peptide, characterized in that Its amino acid sequence is: LTFK c HYWK c QLTSAA, and the side chain amino groups of two lysine residues in the sequence are cross-linked.

2. The stapled peptide according to claim 1, characterized in that The crosslinking group used in the crosslinking is 1,3-dimethylenephenyl.

3. The stapled peptide according to claim 1 or 2, characterized in that The N-terminus of the stapled peptide was acetylated and the C-terminus was amidated.

4. A preparation targeting MDM2 and MDMX proteins, characterized in that: The invention comprises the stapled peptide according to any one of claims 1 to 3.

5. A dual inhibitor of p53-MDM2 / MDMX protein interaction, characterized in that: It comprises the stapled peptide according to any one of claims 1 to 3, or the preparation according to claim 4.

6. The dual inhibitor according to claim 5, characterized in that The dual inhibitor can bind to MDM2 and / or MDMX proteins in cells, activate p53 protein, and exert a tumor suppressor effect.

7. Use of the stapled peptide according to any one of claims 1 to 3, the preparation according to claim 4, or the dual inhibitor according to claim 5 or 6 in the preparation of an inhibitor or drug for treating tumors.

8. The use according to claim 7, characterized in that The tumor is a tumor caused by overexpression of MDM2 and / or MDMX in p53-MDM2 / MDMX.

9. The use according to claim 8, characterized in that The tumor is breast cancer, colorectal cancer or non-small cell lung cancer.

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