Polypeptide and anti-tumor drug containing the polypeptide

Abstract

The present invention relates to the field of nano-drug technology, and particularly to polypeptides and anti-tumor drugs containing polypeptides. The present invention utilizes a three-segment hydrophilic-hydrophobic ARF-mimicking polypeptide to construct a deformable nano-drug delivery system, and simultaneously solves the problems of "gene mutation" and "functional inhibition" of p53. It has been verified that the nano-drug delivery system of the present invention exhibits good biocompatibility and anti-tumor efficiency.

Description

Technical Field

[0001] The present invention relates to the field of nano-drug technology, and particularly to polypeptides and anti-tumor drugs containing polypeptides. Background Art

[0002] Cancer remains one of the greatest threats to human health worldwide, causing over 10 million deaths worldwide each year. For decades, tumors have been classified and studied according to the primary organ, which has benefited several generations. However, cancers from the same organ often have significant heterogeneity, and moreover, cancers from different organs may have many common features. Comprehensive analysis of different types of tumors can greatly improve our ability to predict tumor development. The TP53 gene mutation is considered to be one of the most common alterations in human cancers, so all cancers have similar oncogenic transcriptional signatures.

[0003] The p53 tumor suppressor encoded by the TP53 gene, also known as the guardian of the genome, plays a key role in preventing the occurrence and development of cancer. Although the functional activation of mutant p53 has been regarded as a therapeutic strategy for decades, so far, no p53-based drugs have been approved due to the extremely difficult development of p53 mutant rescue drugs. The introduction of new methods helps to increase the number of ongoing clinical trials of p53-targeted strategies. CP31398 identified by Pfizer in 1999 was the first reported mutant p53 reactivation compound. APR-246, a methylated derivative of PRIMA-1, was the first compound to enter clinical trials and showed strong anti-tumor ability against primary ovarian cancer cells with TP53 missense mutations. In addition, some small molecules, such as MIRA-1 and STIMA-1, can stabilize the wild-type conformation of p53 through their Michael acceptor properties, but did not enter clinical development due to solubility problems and side effects. Arsenic trioxide (ATO; Trisenox), a drug approved for the treatment of acute promyelocytic leukemia, has recently been reported to have the ability to rescue structural p53 mutations. However, the toxicity of these drugs themselves inevitably raises concerns about their translational prospects.

[0004] Another strategy most widely adopted in p53-targeted therapy is to block its inhibitory proteins. Although this strategy is effective almost exclusively in cancer patients with wild-type p53 retained, considering the amplified expression of MDM2 in many cancer types, the development of MDM2-targeted drugs is very promising. MDM2 mainly controls p53 by acting as a ubiquitin ligase (E3), making it a target for p53 degradation. ARF (p19ARF in mice and p14ARF in humans) is a well-known tumor suppressor that can activate p53 by inhibiting MDM2. MDM2 contains several conserved domains, including an N-terminal p53 interaction domain (N-MDM2), a central acidic domain, and a C-terminal RING domain, all of which seem important for the inhibition of p53. For example, nutlin-3 is the most famous compound in anti-cancer research and acts by occupying the p53 binding pocket of MDM2. Under normal circumstances, mammalian ARF is maintained at a low level and is sequestered in the nucleolus due to its C-terminal arginine-rich sequence pattern. Studies on the N-terminus of ARF have shown that the first 22 residues of the N-terminus of ARF play a key role in stabilizing and activating p53.

[0005] Previous studies have shown that in p53 wild-type cancers, the expression of the MDM2 inhibitor ARF is suppressed, which may be the carcinogenic mechanism in patients carrying wild-type p53, namely the p53 inhibition phenomenon. Therefore, restoring the function of ARF has become a potential tumor treatment strategy.

[0006] How to provide an effective solution to the problems of p53 inhibition and p53 mutation has become an urgent technical problem in this field. Summary of the Invention

[0007] To solve the above technical problems, the present invention proposes a p53-targeted cancer therapy strategy based on nanotechnology and ARF mimetic peptides. Specifically,

[0008] First, the present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Using the above three - segment hydrophilic - hydrophobic ARF - mimicking polypeptide, a deformable nano - drug delivery system can be constructed, which can simultaneously solve the problems of "gene mutation" and "function inhibition" of p53. Specifically, the left - hand amino acid residues RVRR in the polypeptide are hydrophilic, and the right - hand amino acid residues KLVFF are hydrophobic. The drug with the function of p53 mutant resurrection can be encapsulated in the center of the nanoparticles through self - assembly. When the nanoparticles reach the action site, the left - hand amino acid residues are cleaved by enzymes in the cells, and the nanoparticles deform and release the encapsulated drug with the function of p53 mutant resurrection, effectively solving the p53 mutation problem. Moreover, the polypeptide sequence in the center of the polypeptide sequence not only acts as a carrier to play a drug - delivery role, but also can inhibit MDM2, effectively solving the p53 inhibition problem.

[0010] In addition, the present invention also optimizes the right - hand amino acid sequence, so that the right - hand amino acid sequence not only has appropriate hydrophobic properties, cooperates with the left - hand amino acid sequence to self - assemble into nanoparticles to achieve drug delivery, but also can promote the inhibition of MDM2 by the middle polypeptide.

[0011] Furthermore, the present invention provides an anti - tumor drug, which contains the above - mentioned polypeptide.

[0012] Preferably, the anti - tumor drug contains the above - mentioned polypeptide and a drug with the function of p53 mutant resurrection.

[0013] Preferably, the drug with the function of p53 mutant resurrection is a hydrophobic drug, more preferably arsenic trioxide.

[0014] Preferably, the weight ratio of the polypeptide to arsenic trioxide is 8 - 9:1.

[0015] Preferably, the particle size of the anti - tumor drug is 30 - 40 nm.

[0016] Furthermore, the present invention provides a preparation method of the above - mentioned anti - tumor drug, including: mixing the above - mentioned polypeptide and the drug with the function of p53 mutant resurrection, and preparing it by the method of thin - film hydration.

[0017] Furthermore, the present invention provides the application of the above - mentioned polypeptide in drug delivery.

[0018] Furthermore, the present invention provides the application of the above - mentioned polypeptide in the preparation of anti - tumor drugs.

[0019] Preferably, the anti - tumor drug is used to regulate the function of MDM2.

[0020] Preferably, the anti - tumor drug is used to regulate the function of the p53 tumor suppressor factor.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention utilizes a three - segment hydrophilic - hydrophobic ARF - mimicking polypeptide to construct a deformable nano - drug delivery system, simultaneously solving the problems of "gene mutation" and "functional inhibition" of p53. It has been verified that the nano - drug delivery system of the present invention exhibits good biocompatibility and anti - tumor efficiency. Brief Description of the Drawings

[0023] Figure 1 It is a test result diagram of the interaction force between different polypeptide sequences designed by the present invention and MDM2 protein.

[0024] Figure 2 It is an electron micrograph of the anti - tumor drug prepared in the embodiment of the present invention.

[0025] Figure 3 It is a particle size distribution diagram of the anti - tumor drug prepared in the embodiment of the present invention.

[0026] Figure 4 It is a test result diagram of the cell survival rate of different cell lines provided by the present invention.

[0027] Figure 5 It is a graph of the change in the body weight of mice provided by the present invention.

[0028] Figure 6 It is a graph of the change in the tumor volume of mice provided by the present invention.

[0029] Figure 7 It is a graph of the change in the survival rate of mice provided by the present invention. Detailed Embodiments

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts fall within the scope of protection of the present invention.

[0031] For those not specifying specific techniques or conditions in the embodiments, they are all conventional methods or are carried out according to the techniques or conditions described in the literature in this field, or according to the product instructions. For those reagents and instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0032] The following embodiments take arsenic trioxide with a structural p53 mutation - resurrecting effect as an example to illustrate the excellent effects of the present invention.

[0033] Example 1 Screening and Synthesis of Polypeptides

[0034] Design different polypeptide sequences against the MDM2 protein, as shown in Table 1, SEQ ID NO.2-8. Test the interaction forces between different polypeptide sequences and the MDM2 protein through molecular dynamics simulation, and the results are as Figure 1 shown.

[0035] Table 1

[0036] SEQ ID Name ARF unit SEQ ID NO.2 ARF22 MVRRFLVTLRIRRACGPPRVRV SEQ ID NO.3 ARF27 MVRRFLVTLRIRRACGPPRVRVFVVHI SEQ ID NO.4 ARF22Scr FVTRGCPRRLVARLIRVMVPRR SEQ ID NO.5 MF24 MVRRFLVTLRIRRACGPPRVRV FF SEQ ID NO.6 MF27 MVRRFLVTLRIRRACGPPRVRV KLVFF SEQ ID NO.7 ML MVRRFLVTLRIRRACGPPRVRV NFGAIL SEQ ID NO.8 MY MVRRFLVTLRIRRACGPPRVRV GNNQQNY

[0037] Among them, MF27 is the precursor of RF31. From Figure 1 It can be seen that different amino acid sequences on the right have an obvious impact on the binding force between the middle amino acid sequence and MDM2. When KLVFF is selected, it can greatly promote the interaction force between the middle polypeptide sequence (ARF22) and MDM2. The binding free energy of MF27 (RF31) and MDM2 is the lowest, and the interaction force is the strongest.

[0038] The solid-phase synthesis method is used for the synthesis of polypeptides. Fmoc-Wang resin is selected as the solid-phase carrier, and the Fmoc / tBu orthogonal protection strategy is used. Various amino acids are connected by amide condensation according to the sequence design to synthesize the peptide chain. Finally, the synthesized peptide chain is cleaved and purified from the resin to obtain the RF31 polypeptide with the specified sequence, and the sequence is as shown in SEQ ID NO.1 (RVRRMVRRFLVTLRIRRACGPPRVRVKLVFF).

[0039] Example 2 Preparation of Antitumor Drugs

[0040] Mix the RF31 peptide and arsenic trioxide (ATO) at a mass ratio of 8.5:1 to obtain ATO@RF31 micelles through thin-film hydration. The specific experimental steps are as follows: Weigh 8.5 mg of RF31 polypeptide and 1 mg of arsenic trioxide and place them in a round-bottom flask, add 1.5 mL of DCM to dissolve them completely, and stir magnetically in the dark for 15 min; Rotate the solution in vacuo to form a film, then add 2 mL of ultrapure water, dissolve it again by ultrasonic bath, and stir magnetically in the dark for 3 h; Filter the solution with an organic filter membrane (0.45 μm), and store the filtrate frozen-dried to form a solid powder in the dark at -20 °C. The transmission electron microscope photograph of the prepared antitumor drug ATO@RF31 is as Figure 2 shown.

[0041] The particle size of nanoparticles in the micelles has an important impact on the distribution of drugs in the body and the aggregation at the tumor site. Therefore, the size of the micelles is tested. Specifically: Weigh 1 mg of the freeze-dried solid powder of ATO@RF31 micelles into an EP tube, add 1 mL of ultrapure water to dissolve it completely (concentration is 1 mg / mL), place it in the sample cell of the particle size potentiometer after ultrasonic treatment for 3 minutes, and measure its particle size with a particle size potentiometer (Malvern, UK, Zetasizer Nano ZS). The results are asFigure 3 as shown

[0042] Furthermore, the encapsulation efficiency and drug loading of ATO were calculated using plasma mass spectrometry data (ICP-MS, PerkinElmer) after sufficient ablation with strong acid. The loading amount of ATO was also confirmed by inductively coupled and energy-dispersive X-ray spectroscopy within a spherical aberration electron microscope. The encapsulation efficiency of arsenic trioxide was measured to be 95%, and the drug loading was 12.3%.

[0043] Test Examples

[0044] The antitumor drugs prepared in the above examples were tested for antitumor effects in a variety of p53 genotype cell lines and mouse tumor-bearing models.

[0045] 1. Cell viability test of p53 gene mutant cell lines

[0046] The specific steps were as follows:

[0047] Cancer cells (obtained from the American Type Culture Collection) were cultured in an incubator at 37°C with 5% carbon dioxide. All cell lines were confirmed to be mycoplasma-free. When the cell confluence reached 70%, an appropriate dose of the drug system was added for treatment, and the cells were harvested after 24 hours for cell viability determination.

[0048] The test results were as Figure 4 shown. The results showed that the antitumor drugs prepared in the above examples had good inhibitory effects on a variety of p53 gene mutant cells.

[0049] 2. Antitumor effect test in mouse tumor-bearing models

[0050] The specific steps were as follows:

[0051] 100 μL of a total of 10 6 SK-BR-3 cells (TP53 mutant) were subcutaneously injected into the right abdomen of 4-week-old male nude mice. The tumor-bearing mice were randomly grouped and administered the corresponding drugs via the tail vein. The calculation formula for tumor volume was: V = π × L × W2 / 6 (V, volume; L, length; W, tumor width). All mice were cultured in a room (22 ± 2°C) with a humidity of 40 - 60% and a light / dark cycle of 12 hours / 12 hours.

[0052] The test results were as Figures 5 - 7 shown. The results showed that the antitumor drugs prepared in the above examples had low toxicity in mice, and the RF31 polypeptide could synergistically inhibit tumor growth with arsenic trioxide.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. An anti-tumor drug, characterized in that, Containing the polypeptide described in claim 1 and a drug having the effect of resurrecting p53 mutations; the drug having the effect of resurrecting p53 mutations is arsenic trioxide.

3. The anti-tumor drug according to claim 2, wherein Its particle size is 30 to 40 nm.

4. The preparation method of the anti-tumor drug according to claim 2, characterized in that, Comprising: Prepared by mixing the polypeptide described in claim 1 and arsenic trioxide and then by means of thin film hydration.

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