阅读说明：本技术 一种靶向HRas蛋白的α螺旋多肽抑制剂及其用途 (HRas protein targeted alpha-helix polypeptide inhibitor and application thereof ) 是由 李子刚 尹丰 覃伟容 廉晨珊 于 2020-07-08 设计创作，主要内容包括：本发明一种靶向HRas蛋白的α螺旋多肽抑制剂,其结构式如下所示：<Image he="188" wi="700" file="DDA0002575402630000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明还提供了上述多肽抑制剂在制备用于靶向HRas蛋白的药物中的用途。本发明的靶向HRas蛋白的α螺旋多肽抑制剂可应用于放疗辐射增敏剂。(The invention relates to an α spiral polypeptide inhibitor targeting HRas protein, which has the structural formula shown as follows: the invention also provides application of the polypeptide inhibitor in preparation of a medicament for targeting HRas protein, and the α helix polypeptide inhibitor for targeting HRas protein can be applied to radiation sensitizer for radiotherapy.)

1. An alpha-helix polypeptide inhibitor targeting HRas protein, characterized in that the structural formula is as follows:

wherein isoD is L-isoaspartic acid and Dap represents 2, 3-diaminopropionic acid.

2. Use of an HRas protein-targeting alpha-helix polypeptide inhibitor according to claim 1 for the manufacture of a medicament for targeting HRas protein.

3. Use of the HRas protein-targeting alpha helix polypeptide inhibitor according to claim 1 for the preparation of a medicament for the treatment of HRas high expressing cervical cancer cell lines.

4. Use of an alpha-helix polypeptide inhibitor targeting HRas protein according to claim 1 for the manufacture of a medicament for increasing the sensitivity of cancer cells to radiotherapy.

5. A pharmaceutical composition comprising an HRas protein-targeting α -helix polypeptide inhibitor of claim 1 and monomethyl auristatin E, wherein said HRas protein-targeting α -helix polypeptide inhibitor is present in said composition at a concentration of 10 μ M and said monomethyl auristatin E is present at a concentration of 0.5 nM.

6. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for targeting HRas proteins.

7. Use of the pharmaceutical composition of claim 1 for the preparation of a medicament for the treatment of a high-HRas-expressing cervical cancer cell line.

8. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for increasing sensitivity of cancer cells to radiation therapy.

Technical Field

The invention belongs to the field of bioengineering, and relates to a polypeptide, in particular to an alpha helix polypeptide inhibitor targeting HRas protein and application thereof.

Background

There are three major therapeutic modalities in the cancer treatment field: radiation therapy, chemotherapy and surgery, radiation therapy being one of the important non-surgical treatments for cancer. The radiotherapy is called as 'invisible scalpel', can keep organs and functions thereof, and achieves the aim of minimally invasive and efficient tumor treatment, for example, for treatment of nasopharyngeal carcinoma, because the nasopharyngeal carcinoma is dissected and located in a special position, and the operation treatment is very difficult, but nasopharyngeal carcinoma cells are sensitive to radioactive rays, the radiotherapy is an ideal treatment mode for treating the nasopharyngeal carcinoma, and early-stage nasopharyngeal carcinoma patients can achieve good cure rate by adopting a radiotherapy strategy. To date, the combined use of radiation therapy and chemotherapy has been widely used in tumor therapy worldwide. However, the radiation resistance produced by tumor cells may impair the effectiveness of radiation therapy. Radiation sensitizers have been shown to be effective in some cancers such as prostate cancer, radiation sensitizers can increase the radiation sensitivity of tumor cells to X-rays and the like, protect normal cells and tissues to the greatest extent possible while killing tumor cells at high efficiency, and various radiation sensitizers such as small molecule drugs and RNA drugs have been developed to enhance antitumor effects. Recently, radiotherapy sensitizers to specific target proteins have attracted a great deal of interest in both academia and industry. Various studies have shown that Ras signaling pathways play an important role in the regulation of radiation resistance. The research shows that rigosetib is a regulator for inhibiting Ras-Raf interaction, can interrupt Ras-Raf-MEK-ERK and PI3K/AKT signal pathway, can be used as a radiosensitizer for cervical cancer radiotherapy, and can inhibit the proliferation of cancer cells through G2/M cell cycle block and other mechanisms. In 2005, Kim et al discovered that Ras-targeting siRNA could down-regulate phosphorylation levels of AKT and ERK kinase (MAPK) levels, reduce cell survival in clonogenic experiments, and show radiosensitization. Therefore, the development of effective inhibitors to block the Ras signaling pathway may be a viable strategy to develop effective radiosensitizers.

Based on the interaction sequence of Ras-Sos interface, Patgiri et al developed an alpha helical polypeptide HBS 3 that targets HRas using a hydrogen bond replacement strategy (HBS), which directly targets HRas protein and successfully down-regulates the phosphorylation cascade downstream of Ras signaling. In 2015, leshciner et al developed an all-carbon "stapled peptide" SAH-SOS1A against wild-type and mutant KRas, which was effective in down-regulating phosphorylation levels of ERK and AKT proteins in the Ras signal downstream phosphorylation cascade and inhibiting growth of cancer cells containing KRas mutant genes. HBS 3 polypeptide and SAH-SOS1A polypeptide are key polypeptide fragments based on Ras-Sos interaction interface, namely, the polypeptide inhibitor of the targeted Ras protein is obtained by simulating the amino acid mutation of alpha H helix (amino acid residue 929-944) on the Sos protein and screening suitable ring-closing positions, and the polypeptide design strategy provides an idea for developing the polypeptide inhibitor of the targeted Ras protein. In 2015, Udadhyaya et al developed a cyclic peptide 9A5 with better cell membrane permeability, which could block Ras-effector interaction and was found in subsequent studies to induce cancer cell apoptosis.

Research has reported that an N-terminal aspartic acid nucleation strategy (TD strategy) is developed by constructing an (i, i +3) lactam bond between 2, 3-diaminopropionic acid (Dap) and L-isoaspartic acid at the N-terminal end of the polypeptide, which can immobilize the polypeptide in an alpha-helical structure, and the polypeptide constructed by this strategy has improved stability of the polypeptide and cell membrane permeability. The polypeptide inhibitor developed at present may have the defects of weak membrane penetrating capability of cell membranes, low binding affinity with HRas and the like, and the development of more efficient Ras inhibitors or Ras-related radiotherapy sensitizers is still needed. The use of N-terminal aspartate nucleation strategy to construct stable alpha helix polypeptides to target Ras proteins is a viable approach.

Methyl auristatin E is called MMAE for short and is a synthetic antitumor drug. MMAE is a potentially potent radiosensitizer of X-ray radiation that increases the sensitivity of tumor cells to radiation, a powerful polypeptide cytotoxin and mitotic suppressive agent that inhibits cell division by blocking tubulin polymerization, derived from a polypeptide known as dolastatin, present in the marine mollusk Dolabella auricularia. MMAE polypeptides and antibody conjugates thereof have shown potent in vitro and in vivo activity against a variety of lymphomas, leukemias and solid tumors in preclinical studies. The MMAE can be used as a positive control for researching a relevant experiment of tumor cells on radiation sensitivity, and meanwhile, in order to verify the potential application prospect of the combination therapy of the MMAE and the polypeptide of the targeted Ras protein, the invention further evaluates the research of the polypeptide for inhibiting the Ras signal pathway and the MMAE combination drug on the aspect of X-ray radiation radiosensitization.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an HRas protein targeted alpha helix polypeptide inhibitor and application thereof, and aims to solve the technical problems that the polypeptide inhibitor in the prior art is weak in cell membrane penetrating capability and low in binding affinity with HRas.

The invention provides an alpha helix polypeptide inhibitor targeting HRas protein, which has a structural formula shown as follows (named as H5 polypeptide):

wherein isoD is L-isoaspartic acid (L-isoaspartic acid), and Dap represents 2, 3-diaminopropionic acid.

Further, the invention also provides application of the HRas protein targeted alpha helix polypeptide inhibitor in preparation of a drug for targeting HRas protein.

Furthermore, the invention also provides application of the HRas protein targeted alpha helix polypeptide inhibitor in preparing a medicament for treating the HRas high-expression cervical cancer cell line.

Furthermore, the invention also provides application of the HRas protein targeted alpha-helix polypeptide inhibitor in preparing a medicament for increasing the sensitivity of cancer cell radiotherapy.

Furthermore, the invention also provides a pharmaceutical composition, which contains the HRas protein targeted alpha-helix polypeptide inhibitor and monomethyl auristatin E, wherein the concentration of the HRas protein targeted alpha-helix polypeptide inhibitor in the composition is 10 mu M, and the concentration of the monomethyl auristatin E is 0.5 nM.

Further, the invention also provides application of the pharmaceutical composition in preparation of a medicament for targeting HRas protein.

Further, the invention also provides application of the pharmaceutical composition in preparing a medicament for treating the HRas high-expression cervical cancer cell line.

Furthermore, the invention also provides application of the pharmaceutical composition in preparing a medicament for increasing the sensitivity of cancer cell radiotherapy.

The invention is based on a key polypeptide sequence on the Sos protein of an interaction pocket in a Ras-Sos compound crystal structure, namely an alpha H helix (amino acid residue 929-944) on the Sos protein, the polypeptide sequence is used as a linear control polypeptide L1 (amino acid residue 929-944: FFGIYLTNILKTEEGN), a series of ring-closing polypeptides are prepared by using an N-terminal aspartic acid nucleation strategy, a fluorescence polarization method is used for detecting the binding affinity and the interaction of a stable polypeptide and the Ras protein, and the killing effect of the polypeptide on cancer cells and the application of the polypeptide in the cancer cell radiotherapy sensitization direction are further evaluated at a cell level.

The invention further discusses the application effect of the combined drug of the polypeptide and the MMAE in the aspect of X-ray radiation sensitization. In the invention, a series of experimental screens are carried out to determine a potential polypeptide H5 which has the advantages of high binding affinity with HRas, strong membrane penetrating capability, good cell activity and the like, and then the killing effect of the drug combination of the polypeptide and MMAE on HeLa cells is further evaluated, and the application of the drug combination of the polypeptide and MMAE on the aspect of radiotherapy sensitization of induced HeLa cells is evaluated by a clone formation experiment.

The invention designs and synthesizes H1-H12 and other polypeptides, the specific polypeptide sequence is shown in Table 1, and the expression mode of the polypeptide sequence is the amino acid sequence from N end to C end. The polypeptide L1 contains the original sequence of the Sos 1. alpha.H-helix (amino acids 929-944: FFGIYLTNILKTEEGN), arginine Arg with positive charge is synthesized at the end of all the polypeptides, in order to further increase the cell membrane permeability of the polypeptides by increasing the positive charge of the polypeptides, thereby enhancing the biological activity of the polypeptides, and Trp is additionally added at the end of all the polypeptides for the purpose of concentration determination of the polypeptides. The polypeptide H1/H2 is based on the sequence of the polypeptide L1, Dap and isoD amino acids are introduced at the N terminal of the polypeptide by using an N terminal nucleation template, stable loop-closing polypeptides are constructed between the Dap and isoD amino acids in an (i, i +3) amido bond loop-closing mode, and a proper loop-closing position is screened. The polypeptides H3 and H4 were designed based on the optimized polypeptide sequence (FEGIYRLELLKAEEAN) previously reported by Patgiri et al, again with an amide bond loop at a different amino acid site at the N-terminus.

For polypeptides H5, H6 and H7, the amino acid residue glutamic acid Glu at position 941 on the polypeptide sequence was replaced with glutamine Gln to further increase the overall positive charge of the polypeptide to improve cell membrane penetration. Other polypeptides based on fluorescence polarization experiments, non-essential hydrophobic residues were selectively replaced (H8-H12) or important amino acid asparagine Asn repeated at positions 943 and 944 to further alter or optimize the polypeptide sequence (H10-H12).

The invention adopts a solid phase synthesis polypeptide technology to synthesize a linear polypeptide sequence, and then uses a method of closing a ring by using terminal aspartic acid to stabilize the alpha helical polypeptide of the target HRas.

Experiments prove that the polypeptide H5 has high binding affinity with HRas, can selectively kill a cancer cell line with high Ras expression, H5 can inhibit a Ras/MAPK signal channel and reduce the phosphorylation level of kinases such as downstream signals ERK and the like, and H5 can also effectively inhibit the proliferation of cancer cells and improve the radiation sensitivity of cervical cancer cells during radiotherapy. Monomethyl Auristatin E (MMAE) is a potential radiation sensitizer for radiotherapy, can be used as a positive control in radiation sensitization research of radiotherapy, and experiments show that the polypeptide H5 and the MMAE can synergistically enhance the apoptosis condition after being used in combination, block cells in the G0/G1 stage, and have more remarkable curative effect than that of the polypeptide H5 or MMAE when being used alone in the radiation sensitization research of radiotherapy. Experiments show that the polypeptide-based Ras inhibitor can be potentially applied to radiation sensitizer for radiotherapy, the polypeptide inhibitor expands the research of conformation constrained peptide in the field of radiotherapy, provides a thought for developing efficient Ras-targeted inhibitor, and the combined administration of the polypeptide and MMAE expands the potential application of combined treatment in the field of radiotherapy.

Compared with the prior art, the invention has remarkable technical progress. The invention proves that the polypeptide can be well combined with HRas protein and inhibit the growth of cell lines such as cervical cancer cells with high Ras expression through experiments such as fluorescence polarization detection, MTT experiment and the like. The polypeptide has the functions of inhibiting the growth of cell lines such as a Ras high-expression cervical cancer cell and the like and increasing the radiation sensitivity of the cervical cancer cell to X-rays, is beneficial to solving the problem of the resistance of the cancer cell to X-ray radiotherapy, and simultaneously widens the application range of conformation-constrained peptides.

Drawings

FIG. 1 the binding affinity of H2-FITC and H5-FITC polypeptides to HRas proteins was determined by fluorescence polarization.

FIG. 2 is a circular dichroism spectrum of polypeptides in purified water.

FIG. 3 is a flow cytometry experiment to determine the membrane penetration ability of FITC-modified polypeptides.

FIG. 4 is a confocal laser microscopy image showing the determination of the membrane penetration ability of FITC-labeled polypeptides H2 and H5 and the approximate localization of the polypeptides in HeLa cells to the cytoplasm or nucleus. .

FIG. 5 shows the serum stability assay of the polypeptide L1H2H 5.

FIG. 6 shows the hemolytic activity of polypeptides H2 and H5.

FIG. 7 the killing ability of the polypeptide L1H2H5 on HeLa, A549, HepG2 and 293T cells was analyzed using the MTT assay.

FIG. 8 is a Control group in which the Control group containing DMSO only was used to analyze the killing ability of HeLa cells by the combination of polypeptides H2, H5, MMAE and H5+ MMAE using MTT assay.

FIG. 9 is a flow chart of the induction of HeLa cells to apoptosis by the combination of polypeptides H2, H5, MMAE and H5+ MMAE.

FIG. 10 is a statistical analysis of the induction of apoptosis in HeLa cells by the combination of polypeptides H2, H5, MMAE and H5+ MMAE. The proportion of cell numbers in the regions of Q3 (representing early apoptotic cells) and Q2 (representing middle and late apoptotic cells) was counted, respectively. The apoptosis experiment is repeated at least three times, and the experimental data are uniformly used as a histogram to analyze the apoptosis induced by the combination of the polypeptides H2, H5, MMAE and H5+ MMAE.

FIG. 11 shows that the combination of polypeptides H2, H5, MMAE and H5+ MMAE blocked HeLa cells in the G2/M phase of the cell cycle.

FIG. 12 is a statistical graph of the cell cycle distribution of polypeptides H2, H5, MMAE and H5+ MMAE in combination on HeLa cells. Compared with the control group, the polypeptide H2, H5, MMAE and H5+ MMAE combined drug group has an increased proportion of cells in the G2/M phase, which indicates that each experimental group can block the cells in the G2/M phase. Data are shown as mean ± sd, repeated at least three independent experiments. The standard deviation is shown as error bars in the figure.

FIG. 13 is a graph investigating the effect of polypeptides on the level of phosphorylation of downstream ERKs in the Ras-Raf-MEK-ERK signaling pathway. (A) Treating HeLa cells with combined drugs of polypeptides H2, H5, MMAE and H5+ MMAE for 24 hours, adding 20ng/ml EGF for stimulation for 10min, extracting cell lysate for western blot experiment; (B) and (3) carrying out semi-quantitative analysis on the result of the western blot experiment, namely analyzing the relative content of pERK/GAPDH.

FIG. 14 shows the clone formation experiment of HeLa cells treated with the combination of polypeptides H2, H5, MMAE and H5+ MMAE after X-ray irradiation. (A) Cloning to form a survival rate curve; adding medicine into cells, incubating for 24h, irradiating with X-rays at different doses, digesting the cells, counting, re-planting the cells in a six-well plate, waiting for 9-11 days to allow the cells to grow clones, dyeing, air drying and counting; a 10% survival score line (SF 10%) is plotted in the figure, while Vehicle is the DMSO control group; (B) clone formation experiments the average lethal dose D0 value for each experimental group. Experimental data was performed by the two-tailed Student's t test using GraphPad Prism 6.0 software. Levels of significance are shown as P <0.05 and P < 0.01. Data are shown as mean ± standard deviation, which is shown as error bars in the figure.

FIG. 15 is a schematic diagram of a polypeptide modified by ring closure for inhibiting Ras-Sos interaction, blocking Ras-related signaling pathway, and increasing sensitivity to cancer cell radiotherapy.

FIG. 16 shows a polypeptide of the present invention H5H-WRR-cyclo (isoDFFDap) -IYLTNILKTQEGN-NH₂HPLC profile of (a).

FIG. 17 shows a polypeptide of the present invention H5H-WRR-cyclo (isoDFFDap) -IYLTNILKTQEGN-NH₂Mass spectrum of (2).

Detailed Description

The invention is further described below with reference to the accompanying drawings.