A reactive oxygen responsive self-assembling short peptide and its application in the preparation of cancer therapeutic drugs

Abstract

The present invention discloses an active oxygen responsive self-assembling short peptide and its application in the preparation of cancer therapeutic drugs, belonging to the field of biomedicine. The self-assembling short peptide of the present invention is coupled with a covalently bound active ingredient, an active oxygen responsive group and a lipophilic molecular fragment, which can form a nanosphere under physiological conditions, the covalently bound active ingredient is wrapped inside the nanosphere, and the polypeptide sequence that recognizes cancer cells is exposed outside the nanosphere. The polypeptide sequence on the nanosphere is enriched in the tumor cell by binding to the integrin molecule overexpressed by the tumor cell. Under the action of active oxygen in the tumor microenvironment, the fragment inside the nanosphere is transformed from hydrophobic to hydrophilic, causing the nanosphere to disassemble, exposing the covalently bound active ingredient inside, and acting on the cysteine ​​residues of the tumor cell, achieving specific selective killing of the tumor cell.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an active oxygen responsive self-assembling short peptide and an application thereof in preparing a cancer therapeutic drug. Background Art

[0002] At present, cancer is a major disease that endangers human life and health, and ranks second in the world as the cause of death. Its main drug treatment method is to control the proliferation of cancer cells through chemotherapy. However, the non-specific recognition of drugs not only kills cancer cells, but also inhibits the growth and reproduction of normal cells, damages normal body functions and immunity, and brings patients a variety of drug side effects, such as nausea, vomiting, anorexia, physical weakness and other symptoms. Therefore, by specifically recognizing the target polypeptide sequence, it is expected to improve the cancer cell selectivity of the effective ingredients, avoid interference with normal cells, improve the effectiveness and specificity of the drug action, and achieve a highly selective killing effect on cancer cells.

[0003] Peptide sequences are the basic structures that constitute biologically active macromolecular proteins. They precisely regulate the interactions of proteins in the body to achieve their biological functions. Peptide sequences have natural recognition properties and excellent biocompatibility for biological targets. Therefore, in modern drug research and development, active peptide fragments are widely used in the development of new and highly effective drugs. Peptide fragments form a variety of nanostructures under different physiological conditions, which in turn affects their interactions with target biomacromolecules. In recent years, with the structural analysis of more and more effective active peptide fragments, artificially designed and synthesized self-assembling peptides have become a research hotspot in the field of biomedicine. Among them, by introducing target-specific recognition sequences and biological microenvironment response fragments to regulate the assembly structure of molecules in self-assembling peptide molecules, it is expected to develop highly effective and specific cancer therapeutics.

[0004] Peptide molecules have special advantages in biomacromolecule target recognition and microenvironment responsiveness. Through reasonable peptide sequence design, sequences containing tumor cell-specific target identification and structural fragments of microenvironment response are introduced into molecules. The resulting new compounds have high and specific recognition of tumor cells, realizing targeted treatment of tumor cells. Integrins are overexpressed in a variety of tumor cells, and they promote the growth, proliferation and migration of tumor cells. At the same time, the expression of reactive oxygen species in the tumor microenvironment is usually significantly higher than that in normal tissues, which is conducive to the destruction of the extracellular matrix of tumor cells and promotes tumor cell proliferation. Covalently bound drug therapeutic molecules show excellent effects in tumor treatment, but their selectivity is poor, which inevitably brings a variety of side effects. If integrins and reactive oxygen species can be used as response factors to design polypeptide sequences, combined with the structural regulation of polypeptide molecules to covalently bind active ingredients and tumor cells, it is expected to prepare self-assembling polypeptide molecules that specifically respond to tumor cells, which can be used to improve tumor cell selectivity, targeting and therapeutic effects.

[0005] Although there are a lot of reports on the use of self-assembling peptides for tumor treatment, the low pharmacological activity seriously restricts the development of this type of drug. Covalently bound drugs have excellent pharmacological activity, but their non-targeted effects seriously limit their widespread application. Therefore, how to obtain a covalently bound molecule that specifically acts on tumor cells is still a problem that researchers in this field need to solve urgently. Summary of the invention

[0006] The purpose of the present invention is to overcome the defect of the non-target effect of existing covalently bound drugs leading to side effects, and to provide a reactive oxygen-responsive self-assembling short peptide containing a covalently bound active ingredient that specifically targets tumor cell integrins and its use in the preparation of cancer therapeutic drugs. The self-assembling short peptide is highly selective for tumor cells, and regulates the interaction between the covalently bound active ingredient and the cysteine ​​residues of tumor cells under the action of reactive oxygen in the tumor microenvironment, thereby selectively killing tumor cells.

[0007] The present invention provides an active oxygen responsive self-assembling short peptide, the structural formula of which is as follows:

[0008]

[0009] Among them, R1 is a covalently bound active ingredient, R2 is an active oxygen response group, R3 is a lipophilic molecular fragment, and n=2-4.

[0010] In some embodiments, the covalently bound active ingredient comprises a molecule comprising the following but not limited to the following structural fragments:

[0011] In some embodiments, the reactive oxygen species responsive group includes molecules containing the following but not limited to the following structural fragments:

[0012]

[0013] In some embodiments, the lipophilic molecule fragments include molecules containing the following but not limited to the following structural fragments:

[0014]

[0015] In some embodiments, the structure of the reactive oxygen responsive self-assembling short peptide is as follows:

[0016]

[0017] The preparation method of the active oxygen responsive self-assembling short peptide includes solid phase and liquid phase synthesis methods.

[0018] The reactive oxygen responsive self-assembling short peptide can self-assemble into nanostructures such as nanospheres and nanocapsules under physiological conditions, covalently bound active ingredients are wrapped inside the nanostructure, and the polypeptide sequence that recognizes the integrin molecule of tumor cells is exposed outside the nanostructure. The physiological conditions are preferably in a phosphate buffer solution environment. The particle size of the nanostructure is preferably 20 to 200 nm.

[0019] The present invention also provides the use of the reactive oxygen responsive self-assembling short peptide in the preparation of cancer therapeutic drugs. The nanostructure formed by the reactive oxygen responsive self-assembling short peptide can be enriched in tumor cells. Under the action of reactive oxygen in the tumor microenvironment, the fragments in the nanostructure change from hydrophobic to hydrophilic, resulting in the disassembly of the nanostructure, exposing the covalently bound active ingredients inside it, and acting on the cysteine ​​residues of tumor cells, achieving specific selective killing of tumor cells, and then achieving the effect of treating cancer.

[0020] The present invention also provides a cancer treatment drug, which comprises the above-mentioned active oxygen responsive self-assembling short peptide and may further comprise pharmaceutically acceptable excipients.

[0021] Compared with the prior art, the present maze has the following advantages and beneficial effects:

[0022] (1) The self-assembling short peptide molecules of the present invention have both integrin targeting and reactive oxygen species response characteristics, and have tumor cell-specific recognition and response properties. The assembly structure can be disassembled under the action of reactive oxygen species.

[0023] (2) In the nanostructure formed by the self-assembled short peptide molecules containing covalently bound active ingredients in the phosphate buffer of the present invention, the covalently bound active ingredients are wrapped inside the nanostructure, which effectively prevents the non-specific effects thereof.

[0024] (3) The self-assembling short peptide molecules containing covalently bound active ingredients of the present invention can achieve targeted recognition of tumor cells and regulate the killing efficacy of active ingredients on tumor cells. Within a specific concentration range (100-400 μM), they can achieve selective killing of tumor cells, have no inhibitory effect on normal cells, and significantly reduce the toxic side effects caused by drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the MS spectrum of intermediate 1.

[0026] Figure 2 is the MS spectrum of intermediate 2.

[0027] Figure 3 is the MS spectrum of compound A1.

[0028] Figure 4The nanostructures of compound A1 and its nanostructures after co-incubation with hydrogen peroxide. (a) Nanostructure of compound A1. (b) Nanostructure of compound A1 after co-incubation with hydrogen peroxide. Scale bar: 100 nm.

[0029] Figure 5 This is the MS spectrum of compound A1 after incubation with hydrogen peroxide.

[0030] Figure 6 This is the MS spectrum of the solution of compound A1 incubated with hydrogen peroxide and further incubated with cysteine.

[0031] Figure 7 The results of the test of the inhibitory activity of compound A1 on different cells. (a) MuM-2B, (b) OCM-1A, (c) HeLa, (d) MDA-MB-231, (e) 293T, (f) the inhibitory activity of compound chlorambucil on 293T cells.

[0032] Figure 8 This is a fluorescence imaging analysis diagram after compound A1 was co-incubated with MuM-2B cells, DAPI (blue), F-actin (green), A1 (red). DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0034] Example 1 Preparation of self-assembling short peptide-covalent binding component coupling molecules

[0035] The coupling molecule representative A1 structure is shown below:

[0036]

[0037] The synthesis method of coupling molecule A1 is as follows:

[0038] (1) Synthesis of intermediate 1

[0039]

[0040] Fmoc-Glu-OtBu (3000 mg, 7.05 mmol), HBTU (5346.7 mg, 14.9 mmol), and DIEA (3.6 mL, 21.2 mmol) were dissolved in 30 mL DCM and stirred at 0°C for 2 h; then thiomorpholine (1.2 mL, 9.63 mmol) was slowly added dropwise to the mixed solution and stirred at 0°C overnight; after confirming that the reaction was complete by TLC (Petro: EtOAc = 1: 1, v / v), the solvent was evaporated at 45°C using a rotary evaporator to obtain an intermediate product; the intermediate product was dissolved in 30 mL DCM, cooled to 0°C in an ice bath, and stirred for 30 min; 6 mL was slowly added in small amounts and multiple times; TFA, stirred at 0°C overnight; after confirming the reaction was complete by TLC (Petro: EtOAc = 1: 1), the solvent was evaporated at 45°C using a rotary evaporator; the product was separated and purified by chromatography column separation, and the solvent was evaporated using a rotary evaporator. The intermediate 1 was obtained as a light yellow solid, which was collected and stored at 4°C. Its mass spectrometry characterization is as follows Figure 1 shown.

[0041] (2) Synthesis of Intermediate 2

[0042]

[0043] 4-Carboxybutyltriphenylphosphonium bromide (3000 mg, 6.77 mmol), HBTU (2600.5 mg, 6.86 mmol), and DIEA (5.9 mL, 33.96 mmol) were dissolved in 30 mL DCM and stirred at 0°C for 1 h. Fmoc-Lys-OH·HCl (2442.7 mg, 6.77 mmol) was slowly added in small amounts and several times, and stirred at 0°C overnight after the addition was complete. TLC (EtOAc: MeOH = 1: 1, v / v) was used to detect the end of the reaction. After evaporation to dryness, the product was separated and purified by chromatography column separation and concentrated by rotary evaporator. The concentrated product was completely dissolved in 7 mL DMF, 150 mL ice water was prepared and placed on a magnetic stirrer, and the dissolved solution was slowly added to the ice water with a rubber-tipped dropper, and the solution turned milky white. TFA was slowly added to the solution to adjust the solution to pH = 3. 50 mL of DCM was poured into a separatory funnel and extracted three times. The lower layer of DCM solution was taken. If the stratification is not obvious, an appropriate amount of saturated salt water can be added to promote stratification; the DCM solution was extracted twice with saturated salt water; excess anhydrous sodium sulfate was added to the DCM solution and allowed to stand for 3 hours; it was filtered and evaporated to dryness on a rotary evaporator. The intermediate 2 obtained was a light yellow solid, which was collected in a ziplock bag and stored at 4°C. Its structure was characterized by mass spectrometry as shown below. Figure 2 shown.

[0044] (3) Synthesis of coupling molecule A1

[0045]

[0046] Step 1: Add 2-chlorotrityl resin (0.5 g, about 0.5 mmol) to the reactor and swell it in anhydrous DCM for 20 min, then dissolve L-Fmoc-Asp(OtBu)-OH (617.9 mg, 1.5 mmol) in anhydrous DMF and add it to the reactor, then add DIEA and react at room temperature for 30 min to couple it to the resin; wash the resin with anhydrous DMF 3 times, block the unreacted sites in the resin with a mixture of DCM / MeOH / DIEA (80:15:5, v / v / v) for 10 min×2 times, then wash it with anhydrous DMF 5 times; add a 20% (v / v) piperidine solution (diluted with DMF) and react at room temperature for 30 min to remove the Fmoc protecting group on the amino acid. Step 2: Dissolve Fmoc-Gly-OH (1.5 mmol) in anhydrous DMF and add it to the reactor, then add DIEA and react at room temperature for 30 min to couple it to the previous amino acid. After the reaction, wash with anhydrous DMF for 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino acid. Step 3: Dissolve Fmoc-Arg(pbf)-OH (1.5mmol) in anhydrous DMF, add to the reactor, and then add DIEA to react at room temperature for 30 minutes to couple it to the previous amino acid. After the reaction, wash with anhydrous DMF for 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino acid. Step 4: Dissolve intermediate 2 (1.5mmol) in anhydrous DMF, add to the reactor, and then add DIEA to react at room temperature for 30 minutes to couple it to the previous amino acid. After the reaction, wash with anhydrous DMF for 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino acid. Step 5: Dissolve Fmoc-Phe-OH (1.5mmol) in anhydrous DMF, add to the reactor, then add DIEA and react at room temperature for 30min to couple it to the previous amino acid. After the reaction, wash with anhydrous DMF 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30min to remove the Fmoc protecting group on the amino acid. Step 6: Dissolve Fmoc-Phe-OH (1.5mmol) in anhydrous DMF, add to the reactor, then add DIEA and react at room temperature for 30min to couple it to the previous amino acid, and react at room temperature for 30min. After the reaction, wash with anhydrous DMF 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30min to remove the Fmoc protecting group on the amino acid. Step 7: Dissolve intermediate 1 (1.5mmol) in anhydrous DMF, add to the reactor, then add DIEA and react at room temperature for 30min to couple it to the previous amino acid.After the reaction, wash with anhydrous DMF 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino acid. Step 8: Dissolve chlorambucil (1.5mmol) in anhydrous DMF, add to the reactor, and then add DIEA to react at room temperature for 30 minutes to couple it to the previous amino acid. After the reaction, wash with anhydrous DMF 5 times; add a 20% piperidine solution (diluted with DMF), react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino acid. 0.1% (v / v) TFA (diluted with DCM) dissociates A1 from the resin for 2h. After removing the solvent, the target compound is purified by chromatography column separation. The obtained product is cut off the protecting group by TFA and washed with ether. Finally, the pure A1 is obtained as a light gray powder, which is sealed and stored at -20°C. Its structural characterization mass spectrum is as follows. Figure 3 shown.

[0047] A phosphate buffer solution of A1 was prepared to a concentration of 0.2 mM. After the solution was left to stand overnight, the nanostructures in the solution were observed using a transmission electron microscope, and it was observed that a nanospherical structure ( Figure 4 a). Add 0.2 mM hydrogen peroxide to the solution that has been standing overnight, incubate in a 37°C water bath for 7 h, and observe using a transmission electron microscope, and the nanosphere structure disappears ( Figure 4 b). LCMS detection of samples before and after adding hydrogen peroxide ( Figure 5 ), it can be seen that A1 produces its oxidized structure under the action of hydrogen peroxide, which increases the hydrophilicity of the compound and leads to the disassembly of the nano-spherical structure. The above solution was further incubated with cysteine ​​and detected by LCMS ( Figure 6 ), it can be found that the covalently bound active ingredient is covalently bound to the sulfhydryl group of cysteine.

[0048] Example 2 Cytotoxicity Experiment

[0049] Digest tumor cells (MuM-2B, OCM-1A, HeLa, and MDA-MB-231) and normal cells (293T) in the logarithmic growth phase, centrifuge at 1200rpm for 3min, and resuspend and count. Inoculate cells into 96-well plates at a density of 5000-8000 cells / well, add 100μL of complete medium to each well, and incubate in an incubator overnight until the cells are completely attached. Discard the upper culture medium, and add 100μL of drugs diluted to different concentrations with complete medium to each well at concentrations of 400μM, 200μM, 100μM, 50μM, 25μM, and 0μM. Operate according to the time gradient of incubation for 72h, 48h, and 24h. After incubation, add 10μL of pre-prepared MTT solution (concentration of 10mg / mL) to each well. After adding MTT for 4 hours, purple needle-shaped crystals were formed. 100 μL of 10% SDS solution was added to each well and placed in an incubator overnight. The corresponding OD value of each well at 570 nm was measured by an ELISA instrument and the data was saved for further data analysis. The formula for calculating cell viability is:

[0050] Cell viability = (OD 治疗组 –OD 空白 ) / (OD 对照组 –OD 空白 )*100%

[0051] Cytotoxicity test results Figure 7As shown, when the concentration of A1 is 400 μM, the cell survival rate can be reduced to 81% after 24 hours of co-incubation with MuM-2B cells; when the co-incubation time of A1 with a concentration of 400 μM and MuM-2B cells is extended, the tumor killing and inhibition effects are further increased to 48 hours and 72 hours, and the tumor cell survival rate can be reduced to 60% and 31%. The compound A1 with a concentration of 400 μM can also reduce the cell survival rate to 61% after 24 hours of co-incubation with low-invasive uveal melanoma cells OCM-1A, and the cell survival rate is reduced to 50% after 48 hours of co-incubation. When the incubation time is extended to 72 hours, the cell survival rate of OCM-1A cells can be reduced to 31%. A1 has a more obvious inhibitory and killing effect on HeLa cells. After HeLa cells are co-incubated with A1 with a concentration of 400 μM for 24 hours, the cell survival rate can be reduced to 69%, and when the incubation time is extended to 48 hours and 72 hours, the cell survival rate will be reduced to 59% and 45%. When A1 at a concentration of 400 μM was co-incubated with MDA-MB-231 cells for 72 hours, the survival rate of MDA-MB-231 cells could be reduced to 30%. Even if the co-incubation time was shortened to 48 hours and 24 hours, the survival rate of MDA-MB-231 cells could be reduced to 41% and 68%, respectively, with significant effects. Compound A1 and the drug chlorambucil were co-incubated with human embryonic kidney cells (293T cells). The experimental results showed that when the incubation time was 72 hours, compound A1 could only reduce the survival rate of 293T cells to 70%, which was significantly lower than its inhibitory effect on tumor cells.

[0052] Example 3 Cell co-localization test

[0053] MuM-2B cells in the logarithmic growth phase were digested and centrifuged and counted. 4 / mL density was inoculated into a 35mm confocal culture dish. After the cells adhered to the wall, A1 was added to the dish at a concentration of 200μM, and the co-incubation time was set to 24h. After the co-incubation, the cells were washed twice with 1×PBS solution and fixed with 4% paraformaldehyde for 15min. Then, they were washed twice with 1×PBS solution, and 1mL of permeabilization solution (1% Triton X-100) was added to each dish, and the membrane was permeabilized for 10min. Then, they were washed twice with 1×PBS solution, and then the phalloidin stain was diluted 200 times with PBS under light-proof conditions, and 150μL was added to each dish; at the same time, Congo red stain was added to locate A1, and staining was performed for 30min. After staining, they were washed twice with 1×PBS solution, and the fluorescence distribution of compound A1 and cytoskeleton was observed using laser confocal microscope.

[0054] result( Figure 8 ) showed that A1 was mainly enriched in the tumor cell skeleton through the mediation of integrin, and its covalently bound active ingredients further enhanced the anchoring of A1 to tumor cells, thereby affecting the growth and reproduction of tumor cells.

[0055] The above is an exemplary description of the present invention. It should be noted that the above embodiments can only further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Without departing from the core of the present invention, any simple deformation, modification or other equivalent substitution without highly creative labor by researchers in the field shall fall within the scope of protection of the present invention.

Claims

1. An active oxygen responsive self-assembling short peptide, characterized in that: The structural formula is as follows: Among them, R1 is a covalently bound active ingredient, R2 is an active oxygen response group, R3 is a lipophilic molecular fragment, and n=2-4.

2. The active oxygen responsive self-assembling short peptide according to claim 1, characterized in that: The covalently bound active ingredients include molecules containing the following but not limited to the following structural fragments:

3. The active oxygen responsive self-assembling short peptide according to claim 1, characterized in that: The reactive oxygen species responsive groups include molecules containing the following but not limited to the following structural fragments:

4. The active oxygen responsive self-assembling short peptide according to claim 1, characterized in that: The lipophilic molecular fragments include molecules containing the following but not limited to the following structural fragments:

5. An active oxygen responsive self-assembling short peptide, characterized in that: The structural formula is as follows:

6. The active oxygen responsive self-assembling short peptide according to any one of claims 1 to 5, characterized in that: The active oxygen responsive self-assembling short peptide can self-assemble to form a nanostructure under physiological conditions.

7. The method for preparing the active oxygen responsive self-assembling short peptide according to any one of claims 1 to 5, characterized in that: Including solid phase synthesis method and liquid phase synthesis method.

8. Use of the reactive oxygen responsive self-assembling short peptide according to any one of claims 1 to 5 in the preparation of cancer therapeutic drugs.

9. A cancer treatment drug, characterized in that: The invention comprises the active oxygen responsive self-assembling short peptide according to any one of claims 1 to 5.

10. The cancer therapeutic drug according to claim 9, characterized in that: Contains pharmaceutically acceptable excipients.

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