[Tyr3] Octreoate-D-configuration oncolytic peptide conjugate, and preparation method and application of fluorescent probe of [Tyr3] Octreoate-D-configuration oncolytic peptide conjugate

Abstract

The invention provides a D-configuration oncolytic peptide and [Tyr3] Octreoate-D-configuration oncolytic peptide conjugate as well as a preparation method and application of a fluorescent probe of the D-configuration oncolytic peptide and [Tyr3] Octreoate-D-configuration oncolytic peptide conjugate, and belongs to the fields of polypeptide preparation and biological medicine. According to the invention, a series of [Tyr3] Octreoate-D-configuration oncolytic peptide conjugates and fluorescent probes thereof are synthesized, and are prepared by a solid-phase polypeptide synthesis method. Experiments prove that the [Tyr3] Octreoate-D-configuration oncolytic peptide conjugate obtained by the invention can significantly improve the stability of the oncolytic peptide and improve the selectivity of the oncolytic peptide to tumor cells, and shows a stronger in-vivo and in-vitro anti-tumor effect. Meanwhile, the D-configuration oncolytic peptide conjugate fluorescent probe prepared by the invention realizes space-time tracing and cell localization of the oncolytic peptide, so that the D-configuration oncolytic peptide conjugate fluorescent probe has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide preparation and biomedicine technology, and specifically relates to a [Tyr 3 ] Preparation method and application of Octreotate-D-configuration oncolytic peptide conjugate and its fluorescent probe. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cancer is a major public health problem worldwide, with high morbidity and mortality. In recent years, tumor immunotherapy has attracted widespread attention. Tumor immunotherapy can reactivate and maintain tumor-immune balance, restore the body's anti-tumor immune response, thereby clearing tumors and preventing tumor regeneration. Oncolytic immunotherapy is one of the means of tumor immunotherapy, which directly kills tumor cells and stimulates the immune system to exert efficient anti-tumor effects, including oncolytic viruses, oncolytic peptides, etc.

[0004] Oncolytic peptides are a new type of polypeptide anticancer drugs, most of which are derived from natural host defense peptides. Oncolytic peptides are generally composed of positively charged amino acids and hydrophobic amino acids. Their amphiphilicity and cationicity make them have good tumor inhibitory effects as a whole. Oncolytic peptides have the characteristics of simple structure, easy synthesis, few side effects, and reduced risk of drug resistance. Unlike traditional anticancer drugs that act on various signal pathways or specific targets, oncolytic peptides target negatively charged membrane components and can directly bind to tumor cell membranes to induce membrane lysis, quickly induce cancer cell death, and then release tumor-specific antigens, which in turn induce systemic anticancer immune responses. By directly killing tumor cells and activating immune responses to indirectly kill tumor tissues, oncolytic peptides can achieve efficient tumor killing and reduce the risk of drug resistance.

[0005] Clip-71 (amino acid sequence: H-KFAKFAKKFAKFAKKFAK-NH2) is a linear amphipathic cationic oncolytic peptide that exhibits killing effects on a variety of tumor cell lines, has good synergy with standard anticancer drugs and can reverse drug resistance. The targeted anticancer peptide EP-100 composed of gonadotropin-releasing hormone and Clip-71 has broad prospects. EP-100 can specifically target cells such as ovarian cancer with high expression of LHRH receptors and produce anti-tumor effects.

[0006] Octreotide derivatives [Tyr 3 ]Octreotate(amino acid sequence: ) is a synthetic octapeptide somatostatin analogue, which has high affinity and specificity for somatostatin receptor 2 (SSTR2) in the G protein-coupled receptor family and is a long-acting specific agonist. [Tyr 3 Octreotate as a targeting peptide 177 Lu-DOTA-[Tyr 3 ]Octreotate peptide drug conjugate (PDC) was approved for marketing in 2018. In the clinical trial research stage, [Tyr 3 Octreotate is also used as a targeting peptide for a variety of PDC drug candidates, loaded with other radionuclides, for the treatment of neuroendocrine tumors, non-small cell lung cancer, and pancreatic islet cell tumors.

[0007] All known organisms on Earth have the characteristic of "uniform chirality". The central law followed by life is based on D-type DNA / RNA and L-type polypeptides / proteins. The 20 amino acids commonly found in nature are all L-type amino acids (Note: glycine has no chirality). Natural polypeptides and proteins are almost all composed of L-type amino acids. Scientists focus their research on polypeptides and proteins on L-type polypeptides and L-type proteins. The corresponding protein degrading enzymes in the human body can effectively recognize polypeptides or proteins of natural configuration and cause them to lose their activity. Therefore, traditional L-configuration polypeptides have problems such as short half-life, easy enzymatic hydrolysis, and poor stability.

[0008] D-type peptides and D-type proteins are composed of D-configuration amino acids. So far, there is no evidence that D-type peptides and D-type proteins have ever appeared in the evolution of life on Earth. Compared with natural L-type peptides and L-type proteins, D-type peptides and D-type proteins composed of D-amino acids have unique structures and functions. Due to stereoselectivity and other reasons, they are difficult to be recognized by the degradation enzyme system in the body. Therefore, they have higher stability and lower immunogenicity under physiological conditions, and retain the original protein binding ability.

[0009] The inventors found that Clip-71 (H-KFAKFAKKFAKFAKKFAK-NH2) is a linear polypeptide composed of L-type amino acids. There are eight lysine residues in the sequence and it is easily degraded by proteases. It has many shortcomings such as poor stability, short half-life, and lack of selectivity for tumor cells, which limits its further clinical application. Summary of the invention

[0010] Clip-71 has the disadvantages of poor stability, weak cell membrane penetration, short half-life and poor tumor tissue targeting that are common in traditional oncolytic peptides.3 ]Octreotate-D-configuration oncolytic peptide conjugates and preparation methods and applications thereof. The present invention synthesizes D-configuration oncolytic peptides and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate. In vitro cytotoxicity experiments showed that the modified conjugate peptide can increase the anti-tumor selectivity of the oncolytic peptide, has extremely high enzymatic stability, and has a long-lasting and efficient proliferation inhibition effect on tumor cells. Cell uptake experiments showed that [Tyr 3 ]Octreotate-D-oncolytic peptide conjugates penetrate the cell membrane and enter the cell faster than D-oncolytic peptides, indicating that the conjugated peptide can achieve precise killing of cancer cells by targeting the SSTR2 receptor, proving the cytotoxicity of the targeting peptide mediated by SSTR2. In vivo anti-tumor experiments showed that [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate can effectively inhibit the growth of tumors in mice and significantly prolong the survival of mice. 3 ]Octreotate-D-oncolytic peptide conjugate introduces the fluorescent group 7-DCCA to achieve the tracing and subcellular localization of the conjugate. 3 ]Octreotate conjugates and their fluorescent probes have good practical application value.

[0011] Specifically, the present invention is implemented through the following technical solutions:

[0012] The first aspect of the present invention provides a D-oncolytic peptide and [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, the D-oncolytic peptide and [Tyr 3 ] The Octreotate-D-configuration oncolytic peptide conjugate comprises the following amino acid residue sequence:

[0013]

[0014] The above-mentioned D-oncolytic peptides and [Tyr 3 Octreotate-D-oncolytic peptide conjugates significantly improve the anti-tumor activity and stability of oncolytic peptides. While destroying the tumor cell membrane, they are not easily degraded by proteases. The conjugates can efficiently enter cells through specific receptors and exert anti-tumor effects. 3 ]Octreotate-D-configuration oncolytic peptide conjugates are covalently linked to achieve intracellular tracing and spatiotemporal monitoring of oncolytic peptides and conjugates.

[0015] The second aspect of the present invention provides the above-mentioned D-configuration oncolytic peptide and [Tyr 3 ] A method for preparing an Octreotate-D-oncolytic peptide conjugate, the method comprising synthesizing a D-oncolytic peptide (such as Figure 1 ), and the solid phase peptide synthesis method to synthesize [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate (such as Figure 2 ), the oncolytic peptide was covalently linked to the targeting peptide using the linker group AEEA, and a disulfide bond was constructed on the resin.

[0016] Specifically, the polypeptide is synthesized using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis (Fmoc-SPPS).

[0017] The third aspect of the present invention provides the above-mentioned D-configuration oncolytic peptide and [Tyr 3 ]Application of Octreotate-D-configuration oncolytic peptide conjugates in the preparation of drugs for preventing and / or treating tumor-related diseases.

[0018] Preferably, the tumor includes solid tumors and non-solid tumors, selected from breast cancer, cervical cancer, endometrial cancer, ovarian cancer, meningioma, head and neck glioma, non-small cell lung cancer, bronchial lung cancer, malignant lymphoma, thyroid cancer, thymic cancer, pancreatic cancer, bladder cancer, urothelial carcinoma, colorectal cancer, prostate cancer, gastric cancer, kidney cancer, liver cancer, skin cancer, melanoma, neuroblastoma, pituitary adenoma, acute myeloid leukemia, acute lymphocytic leukemia, granulocytic leukemia.

[0019] Further preferably, the cancer or tumor is a human cancer or tumor that highly expresses the somatostatin receptor SSTR2.

[0020] The fourth aspect of the present invention provides the above-mentioned D-configuration oncolytic peptide fluorescent probe and [Tyr 3 ] The use of octreotate-D-oncolytic peptide conjugate fluorescent probe in spatiotemporal localization and tracing. The fluorescent probe of the present invention can emit blue fluorescence under ultraviolet light, and can be applied to cell and tissue imaging, and further explore the D-oncolytic peptide and [Tyr 3 ]Study on the anti-tumor activity and mechanism of action of Octreotate-D-configuration oncolytic peptide conjugates at the molecular, cellular, tissue and animal levels.

[0021] The beneficial technical effects of the above technical solution are as follows:

[0022] 1. The D-configuration oncolytic peptide synthesized by the present invention and [Tyr 3]Octreotate-D-configuration oncolytic peptide conjugates improve the inhibitory activity of oncolytic peptides on tumor cells. Among them, the IC 50 (like Fig.16 ) were as follows: in A549 cells, 36.6±6.1μM (QYM-1), 28.5±5.1μM (QYM-2), 71.2±8.4μM (QYM-3), 17.9±1.8μM (QYM-4), 36.6±2.9μM (QYM-5), 21.2±3.6μM (QYM-6), 18.1±2.3μM (QYM-7); in 4T1 cells, 74.6±5.6μM (QYM-1 ), 13.4±2.8μM (QYM-2), 46.1±4.9μM (QYM-3), 14.1±2.9μM (QYM-4), 11.6±2.4μM (QYM-5), 4.4±3.7μ M (QYM-6), 5.8±1.1μM (QYM-7); in A20 cells, 20.9±4.9μM (QYM-1), 3.4±1.1μM (QYM-2), 22.3±1.2μM (QY M-3), 14.3±2.3μM(QYM-4), 7.4±2.8μM(QYM-5), 2.5±0.3μM(QYM-6), 6.2±2.9μM(QYM-7); in Daudi cells, 57.1±2.2μM(QYM-1), 25.2±4.1μM(QYM-2), 35.5±2.7μM(QYM-3), 29.1±0.8μM(QYM-4), 14.9±1. 8μM (QYM-5), 3.0±0.6μM (QYM-6), 3.4±0.3μM (QYM-7); finally, in MCF-7 cells, 52.9±1.2μM (QYM-1), 20.8±2.3μM (QYM-2), 46.1±2.0μM (QYM-3), 11.6±2.1μM (QYM-5), 4.4±1.9μM (QYM-6), which was significantly improved compared with the prototype oncolytic peptide Clip-1. The combined use of QYM-2 and [Tyr 3 ]The inhibitory effect of Octreotate on MCF-7 is significantly weaker than the covalent bond between the two.

[0023] 2. The above technical scheme can improve the stability of oncolytic peptides. Clip-71, developed by predecessors, is an oncolytic peptide composed of L-type amino acids, rich in lysine residues, easily degraded by proteases in the body, with poor stability and short half-life, which limits its wide application. The present invention has carried out a series of structural optimizations, including C / N-terminal modification, amino acid sequence replacement, etc., and uses stable D-configuration amino acids to synthesize new D-configuration oncolytic peptides. D-configuration oncolytic peptides have extremely high resistance to proteolysis, long half-life, can quickly destroy cell membranes, and have a wide range of anti-cancer capabilities.

[0024] 3. The above technical solution can improve the membrane penetration ability of oncolytic peptides in a short time. 3 ]Octreotate and Clip-71 condensation. 3 Octreotate-D-configuration oncolytic peptide conjugates can carry oncolytic peptides in a short period of time, specifically target and recognize SSTR2 receptors on the surface of tumor cells and quickly internalize into cells, greatly improving the selectivity and membrane permeability of oncolytic peptides, thereby allowing a large number of oncolytic peptides to enter cells to exert anti-tumor effects.

[0025] 4. The above technical solution can greatly improve the selectivity of oncolytic peptides. Tumor cells carry a large amount of negative charge on their surface. Oncolytic peptides directly cause cell lysis through electrostatic action, so they have poor selectivity for normal cells and various tumor cells. The expression levels of SSTR2 receptors in tumor tissues and normal tissues are different, so [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugates can aggregate into tumor cells that highly express SSTR2, thereby improving the selectivity of oncolytic peptides for tumor cells and reducing toxic side effects.

[0026] 5. The above technical scheme can significantly improve the anti-tumor activity of oncolytic peptides in mice. 3 ]Octreotate-D-oncolytic peptide conjugates significantly reduced the tumor weight and volume of Balb / c tumor-bearing mice, while [Tyr 3 ] The anti-tumor effect of the Octreotate-D-configuration oncolytic peptide conjugate was more significant. While the weight of the mice remained basically stable, it significantly inhibited tumor growth and significantly prolonged the survival period of the mice.

[0027] In summary, traditional oncolytic peptides have deficiencies such as lack of cell selectivity and poor enzymatic stability, which hinder their clinical application. In order to avoid the above shortcomings, the present invention designed and synthesized a novel [Tyr 3]Octreotate-D-oncolytic peptide conjugate has the advantages of extremely high enzymatic stability, stronger cell selectivity, and more excellent in vitro and in vivo anticancer activity. At the same time, the fluorescent group 7-DCCA is combined with the D-oncolytic peptide and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugates are used together, and emit blue fluorescence after entering the cell, realizing the tracing and spatial level monitoring of the peptide entering the cell, so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 Schematic diagram of the solid phase polypeptide synthesis method of the oncolytic peptide of the present invention;

[0030] Figure 2 For the present invention [Tyr 3 ] Schematic diagram of the solid phase peptide synthesis method of octreotate-oncolytic peptide conjugate;

[0031] Figure 3 The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatogram and mass spectrum of QYM-1 of the present invention;

[0032] Figure 4 The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatogram and mass spectrum of QYM-2 of the present invention;

[0033] Figure 5 The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatogram and mass spectrum of QYM-3 of the present invention;

[0034] Figure 6 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high performance liquid chromatography and mass spectrum of QYM-4 of the present invention;

[0035] Figure 7 The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatogram and mass spectrum of QYM-5 of the present invention;

[0036] Figure 8 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high performance liquid chromatogram and mass spectrum of QYM-6 of the present invention;

[0037] Fig. 9 The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and mass spectrum of QYM-7 of the present invention;

[0038] Fig.10 To evaluate the D-oncolytic peptides and [Tyr 3 ] Octreotate-D-oncolytic peptide conjugates and combined drugs on tumor cell proliferation inhibition rate curve;

[0039] Fig.11 To evaluate the D-oncolytic peptides and [Tyr 3 ]Time-effect curve of the inhibitory effect of Octreotate-D-oncolytic peptide conjugate on 4T1 and MCF-7 tumor cells;

[0040] Fig.12 The L-oncolytic peptide, D-oncolytic peptide and [Tyr 3 ]Stability test curve of Octreotate-D-configuration oncolytic peptide conjugate in human serum;

[0041] Fig.13 The D-configuration oncolytic peptide fluorescent probe of the present invention and [Tyr 3 ] Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe uptake diagram in MCF-7;

[0042] Fig.14 The D-oncolytic peptide of the present invention and [Tyr 3 ] Schematic diagram of the tumor inhibition effect of Octreotate-D-configuration oncolytic peptide conjugate on tumor-bearing mice;

[0043] Fig.15 The D-oncolytic peptide of the present invention and [Tyr 3 ] Octreotate-D-oncolytic peptide conjugate treatment survival curve of tumor-bearing mice, tumor volume and weight line graph, mouse weight curve graph;

[0044] Fig.16 To evaluate the D-oncolytic peptides and [Tyr 3 ] IC of Octreotate-D-configuration oncolytic peptide conjugates and combined drug administration 50 (μM); DETAILED DESCRIPTION

[0045] Below in conjunction with the accompanying drawings and specific embodiments, the specific embodiments of the present invention are described in further detail. In the specific embodiments, each original reagent and raw material all can be purchased. It should be noted that the following detailed description is exemplary and is intended to provide further description of the present invention. Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0046] As mentioned above, the oncolytic peptide Clip-71 is composed of L-amino acids and has disadvantages such as high cytotoxicity, low enzymatic stability, high immunogenicity, and a single administration method, which greatly limits its widespread clinical application.

[0047] In view of this, in a typical embodiment of the present invention, a D-oncolytic peptide and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate, wherein the oncolytic peptide-polypeptide conjugate comprises the following amino acid residue sequence:

[0048]

[0049] The above-mentioned D-oncolytic peptides and [Tyr 3 ]Octreotate-D-oncolytic peptide conjugates greatly improve the enzymatic stability and in vitro and in vivo anti-tumor activity of oncolytic peptides. By taking advantage of the different expression levels of SSTR2 receptors on the tumor surface, targeted entry into tumor cells is achieved and the toxicity to normal cells is reduced, thereby effectively exerting anti-tumor effects. At the same time, the fluorescent group 7-DCCA is combined with the D-oncolytic peptide or [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugates are used together to achieve peptide tracing and spatiotemporal monitoring.

[0050] In another embodiment of the present invention, the above-mentioned D-oncolytic peptide and [Tyr 3 ] A method for synthesizing an Octreotate-D-configuration oncolytic peptide conjugate and a fluorescent probe thereof, the method comprising: synthesizing the oncolytic peptide by solid phase peptide synthesis; connecting the oncolytic peptide with a linker, a fluorescent group, and a targeting peptide by a condensation reaction, and constructing a disulfide bond on a resin.

[0051] Specifically, the polypeptide is synthesized using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis (Fmoc-SPPS).

[0052] Unless otherwise specified, the present invention selects Wang resin (substitution degree of 0.33 mmol / g) to synthesize polypeptides containing a carboxyl terminus; selects Rink-AmideAM resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides containing an amide terminus; and the amino acids used in the synthesis of D-type polypeptides, except for glycine (Gly, G), are all Fmoc-D-configuration amino acids.

[0053] More specifically, the D-oncolytic peptide and [Tyr 3 ] The synthesis method of the Octreotate-D-configuration oncolytic peptide conjugate and the fluorescent probe thereof comprises:

[0054] Step 1, synthesizing a D-configuration oncolytic peptide using a 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis method;

[0055] Step 2, connecting the synthesized D-configuration oncolytic peptide to a linker group and a fluorescent group through a dehydration condensation reaction between amino acids;

[0056] Step 3: Add a solid phase cyclization reagent to the resin to form a disulfide bond through an oxidation reaction.

[0057] Step 4, adding a peptide cleavage agent to the obtained condensation product, cutting the condensation product from the resin, precipitating with glacial ether, and obtaining a crude polypeptide product after centrifugation;

[0058] Step 5: The crude polypeptide product is separated, purified and freeze-dried to obtain D-oncolytic peptide and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate.

[0059] The specific experimental method of step 1 is as follows:

[0060] According to the reaction volume, weigh a certain amount of Rink-AmideAM resin or Wang resin, wash it once with standard method, soak it in 3-4mL analytical pure DMF solvent at room temperature for 1-2h to swell it, pre-activate it, soak it in 3-4mL analytical pure DMF / DCM mixed solvent (3:1, v:v), shake it in a constant temperature shaker at 28℃ for 30min to fully activate it. After activation, remove the Fmoc protecting group, condense it according to the amino acid sequence, and wash it with standard method after all condensation is completed.

[0061] The method for removing the Fmoc protecting group is as follows: at 28° C., the Fmoc protecting group is removed twice using 2-3 mL of a DMF solution containing 20% ​​piperidine, and the removal time is 5 min and 10 min respectively.

[0062] The amino acid condensation reaction method is: dissolve the amino acid and condensation reagent in analytical grade DMF and add them evenly to the resin. Condensate twice at 28°C, and the second condensation reaction time can be slightly extended to ensure sufficient reaction. When Wang resin is used for condensation, the reactant ratio of the first amino acid condensation is amino acid: DIC: Oxyma: DAMP = 3 times equivalent: 6 times equivalent: 3 times equivalent: 0.4 times equivalent (molar ratio), and the reaction time is 6h and 10h; when other amino acids are condensed, the reactant ratio is amino acid: HCTU: DIEA = 3 times equivalent: 2.8 times equivalent: 6 times equivalent (molar ratio), and the reaction time is 30min and 40min. After the two reactions are completed, wash once in a standard manner. The standard washing method is: use DMF and DCM to rinse alternately, and the specific steps are DMF washing twice, DCM washing twice, DMF washing once, DCM washing once, and DMF washing three times to completely remove impurities in the resin, and then use a water pump to drain the residual solvent.

[0063] The specific experimental method of step 2 is:

[0064] The D-configuration oncolytic peptide synthesized in step 1 is condensed with a linker and a fluorescent group. The reactant ratio is AEEA / 7-DCCA:HATU:HOAt:DIEA=3 times equivalents:2.8 times equivalents:3 times equivalents:6 times equivalents (molar ratio).

[0065] The specific experimental method of step 3 is:

[0066] Add the polypeptide solid phase cyclization reagent to the condensation product synthesized in step 2 at a constant temperature of 28°C to remove the Acm protecting group of cysteine ​​and form an intramolecular disulfide bond with the sulfhydryl group. The reaction ratio of the cyclization reagent is resin: Tl(TFA)3: = 3 times equivalent: 2.34 times equivalent (molar ratio). Dissolve thallium trifluoroacetate in a DMF solution containing anisole, ice bath for 5 minutes, and then add it to the resin and react at room temperature for 3 hours.

[0067] The specific experimental method of step 4 is:

[0068] A peptide cleavage reagent was added to the prepared cyclization product. When synthesizing a D-configuration oncolytic peptide, the ratio was TFA:phenol:water:TIPS=88:5:5:2 (v:v:v:v). When synthesizing a targeting peptide containing a disulfide bond, the ratio of the peptide cleavage reagent was TFA:phenol:water:TIPS=88:5:5:2 (v:v:v:v).

[0069] (trifluoroacetic acid): TIPS: water = 95:2.5:2.5 (v:v:v), placed in a shaker at 28 ° C in the dark for 1.5-2h. After the reaction is completed, the reaction solution is flowed into a three-necked bottle through a synthesis tube, and the residual resin is rinsed twice with 0.5mL of TFA. Bubble and concentrate with high-purity nitrogen to remove excess TFA. When the reaction solution is concentrated to 2-3mL, pre-cooled anhydrous ether is added to the concentrate, and the target polypeptide forms a flocculent precipitate in the solution. Centrifuge the suspension for 3min, discard the supernatant, and obtain a crude peptide product. Repeat the precipitation and centrifugation three times, and place the crude product in a fume hood to dry.

[0070] The specific experimental method of step 5 is:

[0071] The crude peptide product obtained in step 4 was added to a mixed solution of acetonitrile and water containing 1‰ TFA, and completely dissolved with the assistance of ultrasound. The obtained peptide was analyzed and identified using analytical RP-HPLC and ESI-MS. The crude peptide solution was freeze-dried by a freeze dryer, and the freeze-dried powder was dissolved and separated and purified using semi-preparative RP-HPLC, and then the target pure peptide was obtained after another round of freeze drying.

[0072] In another embodiment of the present invention, the above-mentioned D-oncolytic peptide and [Tyr 3 ]Application of Octreotate-D-configuration oncolytic peptide conjugates in the preparation of drugs for preventing and / or treating tumor-related diseases and in anti-cancer or anti-tumor drugs.

[0073] In another specific embodiment of the present invention, the present invention can treat solid tumors and non-solid tumors, including breast cancer, cervical cancer, endometrial cancer, ovarian cancer, meningioma, head and neck glioma, non-small cell lung cancer, bronchial lung cancer, thyroid cancer, thymic cancer, pancreatic cancer, bladder cancer, urothelial carcinoma, colorectal cancer, prostate cancer, gastric cancer, kidney cancer, liver cancer, skin cancer, melanoma, neuroblastoma, pituitary adenoma, acute myeloid leukemia, malignant lymphoma, acute lymphocytic leukemia, and granulocytic leukemia.

[0074] In another specific embodiment of the present invention, the treatable cancer or tumor is a cancer or tumor that highly expresses the somatostatin receptor SSTR2.

[0075] In another specific embodiment of the present invention, the above-mentioned D-configuration oncolytic peptide fluorescent probe and [Tyr 3 ] Application of Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe as a tumor cell proliferation inhibitor and / or tracer for non-therapeutic purposes. The "non-therapeutic purpose" is, for example, inhibiting tumor cell proliferation in vitro and promoting tumor cell death.

[0076] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0077] Example 1

[0078] In this example, all target polypeptides were prepared using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis technology (Fmoc-SPPS). Unless otherwise specified, the present invention selects Wang resin (substitution degree of 0.33 mmol / g) to synthesize polypeptides containing carboxyl termini; selects RinkAmideAm resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides containing amide termini; and the amino acids used in the synthesis of D-type polypeptides, except for glycine (Gly, G), are all Fmoc-D-configuration amino acids.

[0079] The scale of synthetic peptides is generally 0.15 mmol. The basic process of synthesizing peptides containing amide termini is as follows: Figure 1 The basic process for synthesizing peptides containing a carboxyl terminus is shown in Figure 2 shown.

[0080] D-configuration oncolytic peptide solid phase synthesis experiment: Take the resin out of the 4°C refrigerator and restore it to room temperature, weigh 400 mg of Rink-AmideAM resin (1 eq, 0.15 mmol), use DMF and DCM standard wash once, soak the resin with analytical pure DMF at room temperature for 1-2 hours to pre-activate the resin. Then use DMF:DCM=1:1 (v:v) to soak at 28°C constant temperature, and shake the activated resin for 0.5-1h. Add 2-3mL 20% piperidine DMF solution (v:v) to the activated resin, react twice, the first reaction for 5min, the second reaction for 10min, to remove the Fmoc protecting group, and wash it standardly for use.

[0081] Dissolve the amino acid and condensation reagent in analytical grade DMF, mix well, and add to the resin. Condensate each amino acid twice to ensure that the amino acid is fully condensed. After the two reactions are completed, wash once with a standard method and pump dry for use. The ratio of the reactants is amino acid: HCTU: DIEA = 3 times equivalent: 2.8 times equivalent: 6 times equivalent (molar ratio).

[0082] [Tyr 3 ]Synthesis of Octreotate-D-configuration oncolytic peptide conjugate: The above-synthesized oncolytic peptide is subjected to condensation reaction with a linker group via an amide bond, and the D-configuration oncolytic peptide synthesized in step 1 is condensed with an AEEA linker group and a fluorescent group. The reactant ratio is AEEA / 7-DCCA:HATU:HOAt:DIEA=3 times equivalents: 2.8 times equivalents: 3 times equivalents: 6 times equivalents (molar ratio).

[0083] Cyclization, peptide cleavage and separation and purification:

[0084] When all amino acids are connected, add thallium trifluoroacetate to the DMF solution containing anisole, ice bath for 5 minutes, add the thallium trifluoroacetate solution to the solid phase peptide synthesis tube, and shake and react at a constant temperature of 28°C for 3 hours. The reaction ratio of the cyclization reagent is resin: Tl(TFA)3: =3 times equivalent: 2.34 times equivalent (molar ratio).

[0085] Add 20% piperidine solution to the cyclization product to remove the Fmoc protecting group and wash once as standard. Wash five times with DCM, drain and remove the residual solvent in the resin. Add peptide cutting reagent and place in a shaker at 28°C for constant temperature shaking reaction for 2-3h. After the reaction is completed, the solution in the peptide synthesis tube flows into a three-necked flask, and the residual peptide is rinsed with 0.5mL TFA, and rinsed twice. Combine the solutions, use high-purity nitrogen to bubble and concentrate, remove excess TFA, and when the reaction solution is concentrated to 2-3mL, add pre-cooled anhydrous ether to the concentrate, and the target peptide forms a flocculent precipitate in the solution. Centrifuge the suspension for 3min, discard the supernatant, and obtain the crude peptide product. Repeat the centrifugation precipitation three times, and place the product in a fume hood to dry.

[0086] The crude peptide product was dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and the crude peptide was analyzed and identified using analytical RP-HPLC and ESI-MS. The identified crude peptide was freeze-dried in a freeze dryer, dissolved, separated and purified using semi-preparative reversed-phase high performance liquid chromatography, and the target pure peptide was obtained by freeze-drying.

[0087] The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and ESI-MS mass spectra of the seven polypeptides prepared by the present invention are as follows: Figure 3-Figure 9 shown.

[0088] CCK-8 assay to detect tumor cell proliferation inhibition experiment:

[0089] A549, 4T1, A20, Daudi and MCF-7 cells in the logarithmic growth phase were selected and the cells were collected and cultured at approximately 1×10 4 The cells were inoculated on a 96-well plate at a density of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM or 100 μM, 25 μM, 6.25 μM, 1.563 μM, 0.391 μM, 0.098 μM) of QYM-1, QYM-2, QYM-3, QYM-4, QYM-5, QYM-6, QYM-7, and [Tyr 3]Octreotate, set up 3 replicate wells for each group, the DMSO content of all groups was ≤0.1%, and continued to culture in the incubator for 24 hours. After the incubation, CCK-8 solution was added to each well to make the final concentration of 5μmol·L -1 After incubation in the incubator for 3-4 hours, the absorbance value was measured at a wavelength of 450 nm using an enzyme reader and the measurement was repeated three times.

[0090] like Fig.10 As shown, the synthesized novel D-configuration oncolytic peptide and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugates have significant anti-tumor activity. The L-configuration prototype peptide QYM-1 is not sensitive to other tumor cells except A20 cells; the prototype targeting peptide [Tyr 3 Octreotate had no inhibitory effect on the proliferation of all tumor cells. The activity of peptide QYM-3 modified by acetylation and hydrazide terminal did not change significantly, while the activity of D-configuration oncolytic peptide QYM-2 was significantly enhanced, indicating that D-configuration peptides have higher anti-tumor activity than L-configuration peptides. 3 Octreotate-D-oncolytic peptide conjugates have a strong killing effect on all tumor cells, and their activity on suspended cells is higher than that on adherent cells. The anti-tumor activity of the non-covalently linked combination is basically equivalent to that of the D-oncolytic peptide, but weaker than the activity of the covalently linked QYM-6 conjugate of the two, indicating that the formation of covalent bonds plays an important role in inhibiting tumor growth.

[0091] 72h time-effect assay for inhibiting tumor cell proliferation:

[0092] 4T1 and MCF-7 cells in the logarithmic growth phase were selected, the cells were collected by centrifugation and the cells were concentrated to approximately 1×10 4 The cells were inoculated at a density of 100 μL / well on a 96-well plate. After incubation in the incubator for 24 hours, the wells with good adhesion were observed and 50 μL of QYM-1, QYM-2, QYM-3, QYM-5 and QYM-6 were added to each well. Three replicate wells were set up in each group to make the final concentration of 10 μM. After the cells were cultured in the incubator for 4 hours, 8 hours, 24 hours, 36 hours, 48 ​​hours and 72 hours, 15 μL of CCK-8 solution was added to each well to make the final concentration of 5 μmol·L -1 , shake well and place in an incubator for 3-4 hours, measure the absorbance at 450nm wavelength, and repeat the measurement three times.

[0093] The results are as follows Fig.11 As shown, [Tyr 3] The Octreotate-D-configuration oncolytic peptide conjugate, QYM-6, has shown strong tumor cell killing ability at 12 hours, with an inhibitory effect of 80%, and it tends to be stable within 12-72 hours, showing a long-lasting anti-tumor effect. The two L-configuration peptides QYM-1 and QYM-3 reached a peak at around 24 hours, but showed a continuous downward trend after 24 hours, which shows that the terminal modification strategy can only slightly increase the anti-tumor activity, but cannot maintain a lasting killing effect. Compared with the L-configuration peptide, the anti-tumor ability of QYM-2 and QYM-5 peptides has been greatly improved, and the inhibitory effect within 24 hours exceeded 50%, but then there is still a decline to varying degrees.

[0094] Serum stability test:

[0095] Take fresh peripheral blood from normal people, centrifuge it, take the supernatant to get serum, and put it in a -80℃ refrigerator for use. Accurately weigh the QYM-1, QYM-2, QYM-3, QYM-5 and QYM-6 polypeptide powders and dissolve them in PBS solution to prepare a 100μM polypeptide solution. Add equal volumes of 10% serum solution and incubate them in a 37℃ constant temperature water bath. Take 50μL of the reaction solution at 0, 4, 8, 24, 36, 48 and 72h of the reaction, add 50μL of acetonitrile and 250μL of deionized water solution (both containing 0.1% TFA) to terminate the reaction. The reaction solution is stored at -20℃ for use. The reaction solution is melted before use, and the supernatant is taken for RP-HPLC analysis after centrifugation.

[0096] The results are as follows Fig.12 As shown in the figure, after 72 hours of incubation, the L-configuration polypeptides QYM-1, QYM-3, and QYM-5 were completely degraded in the serum, while the D-configuration polypeptides QYM-2 and QYM-6 were relatively difficult to hydrolyze, and about 80% and 40% were still present in the serum after 72 hours. The above results indicate that the stability of the D-configuration polypeptides is much higher than that of the L-configuration polypeptides.

[0097] Uptake experiment of SSTR2 high-expressing cell MCF-7:

[0098] MCF-7 cells growing in logarithmic growth were selected, collected by centrifugation, and the cells were plated at approximately 5 × 10 4The cells were inoculated at a density of 100 / well on a 12-well plate and allowed to adhere overnight. After incubation in the incubator for 24 hours, 500 μL of DMEM medium containing 1 μL of SYBR Green I (10000×) staining solution was added to fluorescently label the cell nuclei for 30 minutes. After labeling, 5 μM QYM-4, 5 μM QYM-7 and 5 μM QYM-2 + 5 μM 7-DCCA were added to the well plate for co-incubation. The culture medium was discarded at 10 minutes, 30 minutes, 60 minutes and 90 minutes, and the cells were washed three times with PBS. Then, phenol red-free DMEM medium was added and observed and photographed under a fluorescence microscope.

[0099] The results are as follows Fig.13 As shown in the figure, as time goes by, the fluorescence intensity detected by QYM-4 and QYM-7 gradually increases, indicating that both are taken up by tumor cells. After 10 minutes of incubation, QYM-7 detected moderate blue fluorescence near the cell nucleus, and the ratio of blue to green fluorescence (about 2) increased significantly. After 90 minutes of treatment, bright blue fluorescence was shown around the cell nucleus, indicating that a large amount of hybrid peptides entered the cell. At the same time, almost no blue fluorescence was observed for non-covalently linked QYM-2 and coumarin within 90 minutes, indicating that non-covalently bound oncolytic peptides and coumarin cannot enter tumor cells. The above results show that SSTR2 high-expressing cells MCF-7 are sensitive to [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate has a higher uptake rate.

[0100] In vivo anti-tumor experiment in mice:

[0101] To construct a tumor-bearing mouse model, approximately 10 7 4T1-LUC cells were injected into the right axilla of 6-week-old male Balb / c mice. The mice were randomly divided into 4 groups, 5 mice in each group, and treated with saline, 5 μmol / kg QYM-2, 2.5 μmol / kg QYM-6, and 5 μmol / kg QYM-6, respectively, by intraperitoneal injection every other day for two weeks. During the experiment, the body weight of the mice and the volume of the tumor (volume = length × width) were measured and recorded every two days. 2 ×0.5). After the experiment, all mice were euthanized, and the tumor tissues were removed and weighed.

[0102] The results are as follows Fig.14 and Fig.15As shown in the figure, from the in vitro tumor images, intraperitoneal injection of QYM-2 did not achieve a good tumor inhibition effect, and the tumor volume was only slightly reduced. The QYM-6 group showed a strong inhibitory effect on tumor growth in vivo. The low dose of 2.5μmol / kg showed a trend of tumor weight and volume reduction, while the mice injected with a high dose of 5μmol / kg had obvious tumor necrosis and volume reduction on the 10th day, and the inhibitory effect was significantly stronger than the low dose group.

[0103] QYM-2 and QYM-6 administration significantly prolonged the survival time of mice. All mice in the three treatment groups survived to 30 days. In contrast, the mice in the control group were euthanized within 15 days due to large tumor growth. During the entire experiment, the weight of the mice did not decrease significantly, indicating that the mice tolerated the peptides well at the therapeutic dose.

[0104] Finally, it should be noted that the above is only a preferred implementation case of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned implementation case, it is still possible for a technician in the field to modify the technical solution recorded in the aforementioned implementation case, or to perform equivalent replacement on part of it. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not a limitation on the protection scope of the present invention. A technician in the relevant field should understand that on the basis of the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A class of D-configuration oncolytic peptides and [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate, characterized in that The D-configuration oncolytic peptide and [Tyr 3 ] The Octreotate-D-configuration oncolytic peptide conjugate comprises the following amino acid residue sequence: QYM-2H-kfakfakkfakkfakkfak-NH2 QYM-4 7-DCCA-AEEA-kfakfakkfakfakkfak-NH2 QYM-6 QYM-7 2. The D-oncolytic peptide, D-oncolytic peptide fluorescent probe, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ] A method for preparing an Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe, characterized in that: The preparation method includes a polypeptide synthesis method based on solid phase synthesis, a solid phase condensation reaction of the polypeptide with a linking group and a fluorescent group, and a disulfide bond construction on a resin; Preferably, the polypeptide synthesis method is 9-fluorenylmethoxycarbonyl-based solid phase polypeptide synthesis (Fmoc-SPPS); Preferably, the D-oncolytic peptide, D-oncolytic peptide fluorescent probe, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ] The synthesis method of the Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe is as follows: The peptides were prepared by solid-phase condensation resin and solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl, and then the synthesized peptides were connected with linking groups and fluorescent groups through solid-phase condensation reaction, disulfide bonds were constructed on the resin through oxidation reaction, and pre-cooled peptide cleavage reagents were added to the condensation products to cut off the condensation products from the resin. The products were then separated, purified, freeze-dried, analyzed and identified to obtain D-configuration oncolytic peptides, D-configuration oncolytic peptide fluorescent probes, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe.

3. The D-oncolytic peptide, D-oncolytic peptide fluorescent probe, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ] Application of Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe in the preparation of drugs for preventing and / or treating tumor-related diseases; Preferably, the tumor includes solid tumors and non-solid tumors, selected from breast cancer, cervical cancer, endometrial cancer, ovarian cancer, meningioma, head and neck glioma, non-small cell lung cancer, bronchial lung cancer, lymphoma, thyroid cancer, thymic cancer, pancreatic cancer, bladder cancer, urothelial carcinoma, colorectal cancer, prostate cancer, gastric cancer, kidney cancer, liver cancer, skin cancer, melanoma, neuroblastoma, pituitary adenoma, ganglioneuroblastoma, gastric cancer, ganglioneuroma, papillary thyroid carcinoma, head and neck squamous cell carcinoma, acute myeloid leukemia, acute lymphocytic leukemia, granulocytic leukemia, malignant lymphoma; Further preferably, the cancer or tumor is a cancer or tumor that highly expresses the somatostatin receptor SSTR2.

4. A pharmaceutical composition, characterized in that The composition comprises the D-oncolytic peptide according to claim 1, a D-oncolytic peptide fluorescent probe, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ]Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe.

5. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the polypeptide according to claim 1 and a pharmaceutically acceptable excipient and / or carrier; Preferably, the carrier includes a buffer, a solubilizer, a filler, a disintegrant, a diluent, a binder, a lubricant, an emulsifier, a surfactant, a colorant, a suspending agent and a flavoring agent.

6. The D-configuration oncolytic peptide fluorescent probe according to claim 1, [Tyr 3 ]Octreotate-D-oncolytic peptide conjugate, [Tyr 3 ]Application of Octreotate-D-configuration oncolytic peptide conjugate fluorescent probe as inhibitor or tracer for non-therapeutic purposes.

7. A method for inhibiting tumor cell proliferation and / or migration and invasion in vitro, characterized in that: The method comprises administering the polypeptide of claim 1, the pharmaceutical composition of claim 4 or the pharmaceutical preparation of claim 5 to tumor cells cultured in vitro.

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