Polypeptide, medicine and use

Abstract

The present invention belongs to the field of biochemistry and discloses a polypeptide comprising: (I) an amino acid sequence as shown in SEQ ID No. 1; or (II) an amino acid sequence formed by adding one or more amino acids before or after the amino acid sequence as shown in (I); or (III) a cell-penetrating polypeptide obtained by combining the polypeptide shown in (I) or (II) with a cell-penetrating peptide. The polypeptide significantly inhibits the growth and proliferation of two prostate cancer cell lines (DU145 and PC3), as well as RNA alternative splicing. The present invention also discloses a drug containing the polypeptide and its uses.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a polypeptide, a medicine and uses. Background Art

[0002] Globally, prostate cancer (PCa) is one of the leading health threats to men. In the United States, prostate cancer ranks first and second among male cancers in incidence and mortality, respectively. According to the 2019 estimates of the my country Cancer Society, PCa currently ranks sixth in incidence and seventh in mortality. Importantly, with the improvement of my country's economic and living standards and the increase in life expectancy, the incidence of PCa has been increasing annually, becoming a primary health concern for middle-aged and elderly men in my country. Currently, anti-androgen deprivation therapy (ADT) is a common treatment for PCa besides surgery. It uses androgen synthesis inhibitors (abiraterone) or androgen receptor inhibitors (including bicalutamide, flutamide, and enzalutamide) to interfere with the androgen / AR signaling pathway, thereby inhibiting cancer cell growth and ultimately achieving therapeutic results. While ADT is highly effective in treating early-stage PCa, nearly all patients will relapse within 1-2 years as castration-resistant PCa (CRPC). Currently, the FDA has not approved any effective drug for the treatment of CRPC, and approved PCa drugs are ineffective against CRPC. Currently, CRPC is essentially incurable worldwide, making in-depth research into new PCa drugs urgent and necessary.

[0003] Genome-wide RNA splicing patterns are abnormal during the development and progression of human prostate cancer. RNA alternative splicing events are positively correlated with the cancer's stemness and malignancy. Increased intron retention (IR) events, a form of RNA alternative splicing, is a hallmark of prostate cancer stemness and malignant progression. A study (DOI:10.1038 / s41467-020-15815-7) demonstrated that the spliceosome inhibitor E7107 effectively prevents drug-resistant recurrence and malignant progression in CRPC, suggesting that targeting tumor spliceosome activity is a novel therapeutic strategy for CRPC.

[0004] WO2013130882A1 discloses compositions and methods for treating cell hyperproliferative diseases using PHF5α inhibitors (such as siRNA, shRNA, antisense oligonucleotides, or pharmaceutical compounds). Exemplary cell hyperproliferative diseases that can be treated with the disclosed PHF5α antagonists include cancers, such as glioma, adenocarcinoma, cervical cancer, or prostate cancer;

[0005] Its instructions also record: A method for treating a cell hyperproliferative disease associated with an oncogenic pathway, the method comprising: a) identifying at least one candidate agent, which is a PHF5a antagonist, a U2AF1 antagonist, a DDX1 antagonist, or a combination thereof; b) determining whether a subject suffers from a cell hyperproliferative disease associated with an oncogenic pathway; and c) if the subject suffers from a cell hyperproliferative disease associated with an oncogenic pathway, administering a therapeutically effective amount of a PHF5a antagonist, a U2AF1 antagonist, a DDX1 antagonist, or a combination thereof to the subject in need.

[0006] The instructions also state that the spliceosome inhibitor is sudemycin, spliceostatin, FR901464, pladienolide, E7107, herboxidine, meayamycin, or a derivative or analog thereof;

[0007] At least one candidate agent comprises a polypeptide, a polynucleotide, or a small molecule compound;

[0008] Those skilled in the art have done little research in this regard, and there are even fewer selectable candidates that actually exist in the form of polypeptides.

[0009] Therefore, the technical problem solved in this case is: how to develop a new peptide-based anti-tumor drug. Summary of the Invention

[0010] The object of the present invention is to provide a polypeptide having multiple selectable forms, which can significantly inhibit RNA alternative splicing of prostate cancer cells, as well as the growth and proliferation of two prostate cancer cell lines (DU145 and PC3).

[0011] At the same time, the invention also discloses a preparation method and application of the polypeptide.

[0012] Unless otherwise specified in the present invention: mM represents millimole / L, nM represents nanomole / L, and μM represents micromole / L;

[0013] To achieve the above object, the present invention provides the following technical solution: a polypeptide having:

[0014] (I), the amino acid sequence shown in SEQ ID No. 1;

[0015] or

[0016] (II) an amino acid sequence formed by adding one or more amino acids before or after the amino acid sequence described in (I);

[0017] or

[0018] (III) A cell-penetrating polypeptide obtained by combining the polypeptide shown in (I) or (II) with a cell-penetrating peptide.

[0019] The above polypeptide sequence has at least the following variations:

[0020] 1. Able to combine with self-assembling peptides, such as NapFFKY, GYYF, KLVFFAE (core sequence in amyloid protein Aβ), to develop new peptide drugs.

[0021] 2. Add or replace with non-natural amino acids, such as changing amino acids from L-type to D-type.

[0022] 3. Modify peptide drugs through chemical modification, such as PEG modification to increase drug circulation time; fatty acid (such as C12 or C18) modification to increase stability and utilization.

[0023] 4. The polypeptide drug is delivered by combining it with drug carriers, such as liposomes, microspheres, micelles and hydrogels, to prepare a new drug dosage form.

[0024] Among the above polypeptides, the cell-penetrating peptide is a cell-penetrating peptide that can be retrieved by CPPsite 2.0 (https: / / webs.iiitd.edu.in / raghava / cppsite / stats1.php), such as several classic cell-penetrating peptides: R9 cell-penetrating peptide, TAT cell-penetrating peptide (GRKKRRQRRRPPQ), Penetratin cell-penetrating peptide (RQIKIWFQNRRMKWKK) MAP (KLALKLALKALKAALKLA), Melittin GIGAVLKVLTTGLPALISWIKRKRQQ.

[0025] Penetrating peptides are a class of short peptides that can carry large molecules into cells, and their ability to penetrate the membrane does not rely on classical endocytosis.

[0026] Since cell-penetrating peptides have been widely used in gene therapy, the above-mentioned cell-penetrating peptides are all classic cell-penetrating peptides, and their functions have been verified in several literatures. Therefore, the combination of the above-mentioned cell-penetrating peptides and the polypeptide of this case can carry the core polypeptide into the cell to exert its function, allowing the core polypeptide to exert its function within the cell.

[0027] In the above polypeptide, the polypeptide has an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 3.

[0028] In the above polypeptide, the polypeptide has an amino acid sequence as shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0029] At the same time, the present invention also discloses a medicine comprising any of the above described polypeptides;

[0030] The polypeptide in the drug is one polypeptide or a mixture of multiple polypeptides.

[0031] Among the above-mentioned drugs, the drug is a drug for treating cancer.

[0032] Among the above-mentioned drugs, the drugs are drugs used to treat stage I and stage II non-small cell lung cancer, neuroblastoma, oral squamous cell carcinoma, prostate cancer, colorectal cancer, and malignant melanoma.

[0033] In the above-mentioned medicine, the dosage form of the medicine is injection or oral preparation.

[0034] In the above-mentioned drug, the drug is an aqueous solution or hydrogel in which the polypeptide is dispersed, or the drug is a liposome, microsphere capsule or micelle encapsulated with the polypeptide, or the polypeptide is used as a shell material to form a liposome, microsphere capsule or micelle to form the drug;

[0035] And / or, the oral dosage form is a pill, tablet, capsule or granule.

[0036] Finally, the present invention also discloses the use of any of the above polypeptides as a drug for preparing a cancer treatment drug.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The polypeptide inhibitor designed by the present invention has the function of inhibiting the growth of prostate cancer cells and RNA variable splicing, and enables the polypeptide drug to enter cells and exert its efficacy by binding to the cell-penetrating peptide R9.

[0039] This peptide drug can specifically bind to MATR3, inhibiting its binding to the interacting protein PTBP1, thereby regulating RNA alternative splicing.

[0040] This peptide can significantly inhibit the growth and proliferation of two prostate cancer cell lines (DU145 and PC3). This peptide drug is expected to become an innovative peptide drug for the treatment of various cancers such as prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A The polypeptide sequence and molecular structure diagram of MA of the present invention;

[0042] Figure 1B The polypeptide sequence and molecular structure diagram of RMA of the present invention;

[0043] Figure 2A This is a diagram showing the effect of affecting RNA alternative splicing in prostate cancer cells provided in Example 2 of the present invention;

[0044] Figure 2B This is a diagram showing the effect of affecting RNA alternative splicing in prostate cancer cells provided in Example 2 of the present invention;

[0045] Figure 3 This is a graph showing the growth inhibition of DU145 cells by some of the polypeptides provided in Example 3 of the present invention;

[0046] Figure 4 This is a graph showing the growth inhibition of PC3 cells by some of the polypeptides provided in Example 3 of the present invention;

[0047] Figure 5 This is a graph showing the growth inhibition of DU145 cells by some of the polypeptides provided in Example 3 of the present invention;

[0048] Figure 6 This is a graph showing the growth inhibition of PC3 cells by some of the polypeptides provided in Example 3 of the present invention;

[0049] Figure 7 These are microscopic photos of the growth inhibition of DU145 cells and PC3 cells by some of the polypeptides provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] Example 1

[0052] Synthesis of peptide drug molecules by solid phase synthesis

[0053] Using AM resin, the desired amino acids are weighed and dissolved in a synthesis flask. Synthesis is performed according to the settings of the fully automated synthesis instrument. The resin is removed, cut, and precipitated to obtain the crude peptide. Purification and lyophilization are then performed using high-performance liquid chromatography to obtain the corresponding peptide drug lyophilized powder.

[0054] The importance of 7aa and 11aa lies in: 7aa is the core active peptide, and 7aa can bind to the MATR3 protein in vitro, but without the membrane-penetrating peptide, it cannot fully enter the cell to exert its effect. Therefore, 7aa, 11aa, and MA will have greater application value after being combined with the membrane-penetrating peptide in the actual treatment process.

[0055] The sequences of the polypeptides synthesized in this example can be found in Table 1:

[0056] Table 1 Sequence Listing

[0057] serial number name sequence SEQ ID No 1 R9 RRRRRRRRR 2 7aa GILGPPP 1 3 11aa PAPGILGPPPP 2 4 MA QSTNPAPGILGPPPPSFHLG 3 5 R9-7aa RRRRRRRRRGILGPPP 4 6 R9-11aa-WT RRRRRRRRRPAPGILGPPPP 5 7 RMA RRRRRRRRRQSTNPAPGILGPPPPSFHLG 6 8 R9-11aa-Mut RRRRRRRRRPAPGAAAPPAP 7

[0058] The structural diagrams of MA and RMA can be found in Figure 1A and Figure 1B .

[0059] Example 2

[0060] Influencing RNA alternative splicing in prostate cancer cells.

[0061] Determination of alternative splicing of cancer cell RNA: The polypeptides R9 and RMA were prepared into a 50mM stock solution with DMSO, diluted to 80μM with culture medium, and added to a 6-well plate with PC3 and DU145 cells pre-plated, 2mL of culture medium per well, and three replicates were set up. After 20 hours, total RNA of the cells was extracted and reverse transcribed into cDNA. The alternative splicing of the target gene was detected by semi-quantitative PCR, and the semi-quantitative PCR products were finally detected by agarose gel. Refer to Figure 1. The results show that RMA can significantly affect the alternative splicing of RNA.

[0062] refer to Figure 2A and Figure 2B , Figure 2A The left side in the middle is R9, and the right side is RMA treatment, which detected the occurrence of alternative splicing of ZMYND8. Figure 2B The left side of the middle image was processed with R9, and the right side with RMA, and the occurrence of variable splicing of ST7 was detected.

[0063] MATR-3 has been reported to act as a splicing factor in prostate cancer to promote cancer development and metastasis. ZMYND8 and ST7 are molecules that have been reported to be affected by MATR-3 splicing. Therefore, using R9 as a control in prostate cancer cells, it was found that RMA can significantly inhibit RNA alternative splicing regulated by MATR-3.

[0064] Example 3

[0065] It has an inhibitory effect on the proliferation of prostate cancer cells PC3 and DU145.

[0066] refer to Figures 3 to 6 Peptides R9, MA, RMA, R9-7aa, R9-11aa-WT, and R9-11aa-Mut were prepared in DMSO to a 50 mM stock solution, diluted to 80 μM in culture medium, and 100 μL of the diluted peptide solution was added to a 96-well plate pre-seeded with PC3 and DU145 cells. Each concentration was replicated in triplicate. After 72 hours of treatment, OD values ​​were measured at 450 nm using CCK-8, and the obtained values ​​were used to calculate cell viability.

[0067] Figures 3 to 6 In the figure, the horizontal axis is the polypeptide concentration; the vertical axis is the cell survival rate.

[0068] refer to Figure 3 , Figure 3 In the experiment, MA did not show any significant inhibitory effect on the growth and proliferation of DU145 cells; RMA showed a significant inhibitory effect on the growth and proliferation of DU145 cells;

[0069] pass Figure 3 It can be shown that MA cannot quickly and significantly inhibit DU145 cells without the assistance of cell-penetrating peptides.

[0070] refer to Figure 4 , Figure 4 In the experiment, MA showed a significant inhibitory effect on the growth and proliferation of PC3 cells; RMA showed an even more significant inhibitory effect on the growth and proliferation of PC3 cells; however, the inhibitory speed of MA was significantly slower than that of RMA;

[0071] pass Figure 3 and 4 It can be proved that MA can inhibit the growth and proliferation of some tumor cells even in the absence of the action of cell-penetrating peptides; in the presence of cell-penetrating peptides, the inhibitory effect is more significant and rapid.

[0072] refer to Figure 5 , Figure 5 R9-11aa-Mut did not show an inhibitory effect on the growth and proliferation of DU145 cells; R9-7aa did not show a significant inhibitory effect on the growth and proliferation of DU145 cells; R9-11aa-WT and RMA showed a significant and rapid inhibitory effect on the growth and proliferation of DU145 cells; the effect of RMA was better than that of R9-11a-WT.

[0073] refer to Figure 6 , Figure 6 R9-11aa-Mut did not show the inhibitory effect on the growth and proliferation of PC3 cells; R9-7aa showed a relatively obvious inhibitory effect on the growth and proliferation of DU145 cells, but the inhibitory speed was not as fast as R9-11aa-WT and RMA; R9-11aa-WT and RMA showed obvious and rapid inhibitory effects on the growth and proliferation of DU145 cells; there was no obvious difference in the inhibitory effect between RMA and R9-11aa-WT, and the inhibitory speed of R9-11aa-WT was slower than that of RMA.

[0074] In summary, the inhibitory effect on the growth and proliferation of tumor cells is exerted by the core polypeptides 7aa, 11aa, and MA. From the perspective of polypeptide function optimization, the core polypeptide MA is the most preferred; when the core polypeptide is combined with the membrane-penetrating peptide, it has obvious advantages in inhibitory effect and speed.

[0075] If more amino acid changes are made to the core polypeptide, such as R9-11aa-Mut, the corresponding effect will disappear.

[0076] This project verified the binding of other transmembrane peptides (TAT, Penetratin) to the core peptide, which was consistent with the trend shown in Example 3.

[0077] Example 4

[0078] For colony formation experiments, growing PC3 and DU145 cells were digested with 0.25% trypsin and counted. 6,000 cells were plated per well of a six-well plate, followed by 2 ml of complete culture medium and continued incubation for 24 hours. R9, MA, and RMA were added to each well at 80 μM, respectively. After 7 days of culture, the medium was discarded, the cells were washed, stained with crystal violet, and microscopically photographed to count the cells.

[0079] The results showed that MA and MA combined with cell-penetrating peptides had a good effect in inhibiting the growth of cancer cells, but the efficacy needs to be further optimized and has certain research and application value.

[0080] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A drug, characterized in that A drug containing a polypeptide according to any one of SEQ ID No. 3 to SEQ ID No. 6; The medicine is a medicine for treating prostate cancer.

2. The drug according to claim 1, characterized in that The dosage form of the medicine is injection or oral preparation.

3. The drug according to claim 1, characterized in that The drug is an aqueous solution or hydrogel in which the polypeptide is dispersed, or the drug is a liposome, microsphere capsule or micelle encapsulating the polypeptide, or the polypeptide is used as a shell material to form a liposome, microsphere capsule or micelle to form the drug; and / or the oral agent is a pill, tablet, capsule or granule.

4. Use of the polypeptide described in any one of SEQ ID No. 3 to SEQ ID No. 6 as a drug for treating prostate cancer.

Images