CN114053427A - Polypeptide targeted drug and preparation method and application thereof - Google Patents

Polypeptide targeted drug and preparation method and application thereof Download PDF

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CN114053427A
CN114053427A CN202111286942.7A CN202111286942A CN114053427A CN 114053427 A CN114053427 A CN 114053427A CN 202111286942 A CN202111286942 A CN 202111286942A CN 114053427 A CN114053427 A CN 114053427A
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polypeptide
protein
peptide segment
targeted drug
cell
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CN114053427B (en
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王浩
王曼迪
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National Center for Nanosccience and Technology China
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

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Abstract

The invention provides a polypeptide targeted drug, a preparation method and application thereof. The polypeptide targeted drug comprises a cell-penetrating peptide segment and a self-assembly recognition peptide segment for recognizing homologous protein, wherein the self-assembly recognition peptide segment is a beta-sheet peptide segment in a protein structure, the sequence of the beta-sheet peptide segment comprises any one of amino acid sequences shown as SEQ ID NO. 1-14, and the C end of the cell-penetrating peptide segment is connected with the N end of the self-assembly recognition peptide segment by an amido bond. The polypeptide targeted drug is prepared by a solid-phase synthesis method, and the method is simple. The polypeptide-targeted drug can induce excessive autophagy death of cells, can be applied to preparation of antitumor drugs, and has important practical significance for clinical treatment of tumors.

Description

Polypeptide targeted drug and preparation method and application thereof
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a polypeptide targeted drug and a preparation method and application thereof.
Background
Tumors are genotypic diseases, and the occurrence, development and metastasis of tumors are closely related to the abnormality of oncogenes. Oncogenes are genes in the cellular genetic organization that can transform normal cells maliciously, and when they are structurally mutated or abnormally expressed, they can induce normal cells to transform, resulting in malignant growth. Traditional antitumor drugs, such as doxorubicin, paclitaxel, cyclophosphamide, raltitrexed and the like, generally have strong cytotoxicity, and although the traditional antitumor drugs can kill tumor cells strongly, the traditional antitumor drugs can also damage normal tissues and cells of a human body. Currently, in clinical treatment of tumors, targeted drug therapy is widely applied due to the advantages of high specificity and small side effect. Among them, the polypeptide drugs have the advantages of low immunogenicity, high receptor binding rate and low cost compared with other small molecule drugs, and are widely concerned by the researchers.
CN109942688A discloses synthesis and application of a polypeptide drug which is combined with Sox2 protein in a targeted manner. The polypeptide medicine combined with the Sox2 protein in a targeted manner is prepared by a chemical synthesis method, the polypeptide medicine combined with the Sox2 protein in a targeted manner is added into a cell culture medium of an esophageal squamous carcinoma cell KYSE450, and the functions of the polypeptide medicine are identified by a cell proliferation experiment, a scratch, a cell invasion and a tumor formation experiment.
CN107952080A discloses a tumor targeting polypeptide-drug conjugate derivative, a preparation method and an application thereof, wherein the polypeptide-drug conjugate derivative comprises a targeting polypeptide, a long-acting polypeptide and a drug molecule, the targeting polypeptide is connected with the C end of the long-acting polypeptide to form a fusion peptide, the fusion peptide is used as a carrier of the drug molecule, and the long-acting polypeptide is a polypeptide or a protein structural domain with affinity with human serum albumin. The polypeptide-drug conjugate derivative has long retention half-life in a blood circulation system, can target and permeate into tumor tissues, releases free effector molecules and exerts antitumor efficacy.
CN112402622A discloses an anti-tumor polypeptide nano-drug carrier targeting PD-L1 and application thereof. The anti-tumor polypeptide nano-drug carrier can activate the immune reaction of T cells, and comprises amphiphilic anti-tumor polypeptide, stearic acid coupled with a polypeptide side chain and enzyme-responsive functional polypeptide. The polypeptide nano-drug carrier has the advantages of simple preparation method, low cost and strong practicability, has stronger tumor inhibition effect compared with single polypeptide, and simultaneously has the advantages of strong tumor part protease responsiveness, good biocompatibility and high stability.
Based on the above studies, it can be seen that the targeted polypeptide drug can effectively inhibit the growth of tumor cells by combining with the target. However, at present, many tumor cells have no clear target, so that the development of novel target protein and the development of corresponding targeted polypeptide drugs become the key point of tumor drug development, and have great practical significance for clinical treatment.
Disclosure of Invention
Aiming at the defects of the prior art and the practical needs, the invention aims to provide a polypeptide targeted medicament and a preparation method and application thereof. The polypeptide targeted drug can be specifically aggregated at a targeted site to induce excessive autophagy death of tumor cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a polypeptide-targeted drug, which comprises a cell-penetrating peptide segment and a self-assembly recognition peptide segment for recognizing homologous proteins.
In the invention, the schematic diagram of the polypeptide-targeted drug is shown in figure 1, and the polypeptide-targeted drug comprises a cell-penetrating peptide segment and a self-assembly recognition peptide segment for recognizing homologous proteins. The schematic diagram of the polypeptide targeting drug entering the tumor cell is shown in fig. 2, the self-assembly recognition peptide segment enters the tumor cell and can recognize homologous protein and aggregate with the homologous protein, so that the protein is denatured, and excessive autophagy death of the cell is induced. The polypeptide targeted drug has the advantages of high biocompatibility, good solubility and strong specificity, and has rapid action, short required time and wide application prospect in the field of tumor targeted therapy.
The self-assembly recognition peptide segment is a beta-sheet peptide segment in a protein structure, the sequence of the beta-sheet peptide segment comprises any one of amino acid sequences shown as SEQ ID NO. 1-14, and the specific sequence is as follows:
SEQ ID NO:1：β1(IFVQGL)；
SEQ ID NO:2：β2(INLYT)；
SEQ ID NO:3：β3(EATVSF)；
SEQ ID NO:4：β4(IKVSFA)；
SEQ ID NO:5：β5(TEFQL)；
SEQ ID NO:6：β6(LVYVHHLGE)；
SEQ ID NO:7：β7(QVYEAT)；
SEQ ID NO:8：β8(FVLKVQ)；
SEQ ID NO:9：β9(SVLVG)；
SEQ ID NO:10：β10(FYSAHL)；
SEQ ID NO:11：β11(TFE)；
SEQ ID NO:12：β12(KLVFFAE)；
SEQ ID NO:13：β13(GNNQQNY)；
SEQ ID NO:14：β14(VQIINK)。
preferably, the cell-penetrating peptide segment and the self-assembly recognition peptide segment are connected by an amido bond.
In the invention, the C end of the cell-penetrating peptide segment is connected with the N end of the self-assembly recognition peptide segment by an amido bond.
Preferably, the sequence of the self-assembly recognition peptide fragment comprises an amino acid sequence shown as SEQ ID NO. 4.
The amino acid sequence shown in SEQ ID NO. 4 is preferably the sequence of the self-assembly recognition peptide segment, can be assembled into regular nano fibers in vitro and has strong ThT response.
In the invention, the sequence of the cell-penetrating peptide segment comprises any one of amino acid sequences shown as SEQ ID NO:15-21, and the specific sequence is shown as follows:
SEQ ID NO:15：TAT(RRRQRRKKRGY)；
SEQ ID NO:16：NLS(SPKKKRKV)；
SEQ ID NO:17：RQIKIWFQNRRMKWKK；
SEQ ID NO:18：VKRGLKLRHVRPRVTRMDV；
SEQ ID NO:19：LLIILRRRIRKQAHAHSK；
SEQ ID NO:20：KLALKLALKALKAALKLA；
SEQ ID NO:21：PFVYLI。
preferably, the sequence of the cell-penetrating peptide segment comprises an amino acid sequence shown as SEQ ID NO. 15.
In the invention, the amino acid sequence shown in SEQ ID NO. 15 is preferably the sequence of the cell-penetrating peptide segment, can penetrate through a cell membrane to enter cytoplasm or even nucleus of a cell, and has NO toxic or side effect on the cell.
In the present invention, the homologous protein includes a cytoplasmic protein or a nuclear protein.
Preferably, the homologous protein comprises any one of FUS protein, Bub-1 protein, TDP-43 protein, K-Ras protein or Myc protein, and is preferably FUS protein.
In the invention, the homologous protein is preferably FUS protein, can form local high-concentration liquid drops in a cell nucleus through liquid-liquid phase separation, and is more favorable for triggered assembly.
In the invention, the N end of the polypeptide targeted drug is connected with a hydrophobic small molecule.
Preferably, the hydrophobic small molecule includes any one or a combination of at least two of NBD, Nap, porphyrin or benzoic acid, for example, the combination may be a combination of NBD and Nap or a combination of Nap and porphyrin, and any other combination may be selected, which is not described herein again.
According to the invention, the N end of the polypeptide targeted drug is connected with the hydrophobic micromolecule, so that the self-assembly performance of the polypeptide targeted drug can be enhanced, and the drug effect of the polypeptide targeted drug is enhanced.
In a second aspect, the present invention provides a method for preparing the polypeptide-targeted drug according to the first aspect, wherein the method for preparing the polypeptide-targeted drug comprises a solid phase synthesis method.
Illustratively, the solid-phase synthesis method may employ a preparation method comprising the steps of:
(1) weighing resin, putting the resin into a polypeptide solid phase synthesis tube, sequentially adding a swelling agent and a deprotection agent, uniformly mixing, washing, taking out the resin, adding a detection liquid to detect the resin to be positive, and respectively adding next amino acid according to the sequences of the cell-penetrating peptide segment and the self-assembly recognition peptide segment to perform amino acid condensation reaction;
(2) respectively selecting amino acids according to the sequences of the membrane-penetrating peptide segment and the self-assembly recognition peptide segment, mixing the amino acids with a condensing agent, adding a reaction solution for dissolving, putting the mixture into a polypeptide solid phase synthesis tube, taking out resin after the reaction is finished, adding a detection solution, washing after the detection shows negative, obtaining peptide resin condensing the first amino acid, repeating deprotection-amino acid condensation reaction until the last amino acid is coupled and deprotected, and washing, concentrating and drying in sequence to respectively obtain the synthesized membrane-penetrating peptide resin and the self-assembly recognition peptide resin;
(3) respectively putting the cell-penetrating peptide resin and the self-assembly recognition peptide resin in the step (2) into a lysate for cracking, and removing impurities and purifying to obtain a cell-penetrating peptide segment and a self-assembly recognition peptide segment;
(4) and (4) connecting the cell-penetrating peptide segment obtained in the step (3) with the self-assembly recognition peptide segment by adopting an amide bond to obtain the polypeptide targeted drug.
In a third aspect, the present invention provides an application of the polypeptide-targeted drug of the first aspect in preparing an anti-tumor drug.
In a fourth aspect, the present invention provides a method of inducing autophagy death in a cell, the method comprising: co-culturing the polypeptide-targeted drug of the first aspect with a cell.
Preferably, the concentration of the polypeptide-targeting drug is 10 to 100. mu.M, and may be, for example, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 60. mu.M, 70. mu.M, 80. mu.M, 90. mu.M, or 100. mu.M, but not limited to the above-mentioned values, and other values not listed in the range are also applicable, and preferably 90 to 100. mu.M.
Preferably, the co-cultivation time is 0.5 to 24 hours, for example, 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc., but the co-cultivation time is not limited to the recited values, and other values not recited in the range are also applicable, preferably 3 to 5 hours.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a polypeptide targeted drug, which comprises a membrane-penetrating peptide segment and a self-assembly recognition peptide segment for recognizing homologous protein, wherein the self-assembly recognition peptide segment can be specifically combined with FUS protein and has a targeting effect, and meanwhile, the self-assembly recognition peptide segment is a first targeted peptide segment with the FUS protein as a target spot;
(2) the self-assembly recognition peptide segment in the polypeptide targeting drug can recognize homologous protein and aggregate with the homologous protein, so that the protein is denatured, and excessive autophagy death of cells is induced;
(3) the polypeptide targeted drug can be used for preparing anti-tumor drugs and has great significance for clinical treatment of tumors.
Drawings
FIG. 1 is a schematic diagram of a polypeptide-targeted drug to which the present invention relates;
FIG. 2 is a schematic diagram of the action of the polypeptide targeting drug into cells according to the present invention;
FIG. 3 is beta4The molecular structural formula of (1);
FIG. 4 is beta4SPRi plots of bound FUS proteins;
FIG. 5 is beta4-TAT;
FIG. 6 is β4-SPRi plot of TAT binding FUS protein;
FIG. 7 is a confocal image of 10. mu.M in vitro recombinant protein FUS-TagRFP-MBP-His;
FIG. 8 shows that 10. mu.M of the in vitro recombinant protein FUS-TagRFP-MBP-His is β -modified4-confocal images after induction of aggregation by TAT molecules;
FIG. 9 is β4-TAT is added to SKBr-3 cells expressing FUS-TagRFP protein, and the co-focusing of the aggregation of FUS-TagRFP protein is carried out over timeAn image;
FIG. 10 is β4Immunofluorescence co-localization confocal image of-TAT-NBD and FUS protein (a is immunofluorescence localization confocal image of cell nucleus, and b is beta)4-immunofluorescence co-localized confocal images of TAT-NBD, c is an immunofluorescence co-localized confocal image of FUS protein, d is a superimposed immunofluorescence co-localized confocal image of a-c);
FIG. 11 is β4-TAT, SKBr-3 and HUVEC cells, and then CCK-8 cytotoxicity detection results are shown;
FIG. 12 is β4-TAT-NBD, Skbr-3 and HUVEC cells after co-incubation, and CCK-8 cytotoxicity detection results;
FIG. 13 is β4A Western blot cytotoxicity detection result chart of-TAT induced SKBr-3 cell autophagy;
FIG. 14 is β4A Western blot cytotoxicity detection result chart of-TAT-NBD induced SkBr-3 cell autophagy;
FIG. 15 is β3-TAT;
FIG. 16 is β3-TAT, SKBr-3 and HUVEC cells, and then CCK-8 cytotoxicity detection results are shown;
FIG. 17 is β3-TAT-NBD, Skbr-3 and HUVEC cells after co-incubation, and CCK-8 cytotoxicity detection results;
FIG. 18 is β10-TAT;
FIG. 19 is β10-TAT and U-2-OS and SH-SY5Y cells after incubation CCK-8 cytotoxicity detection result chart.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the following examples, the formulation of the Fmoc deprotecting agent is piperidine-dimethylformamide ═ 1:4 (v/v); the formula of the reaction solution is nicotinamide mononucleotide, namely dimethylformamide (1: 24) (v/v); the formula of the lysis solution is 95 vol% TFA +2.5 vol% TIS +2.5 vol% H2O; the ninhydrin detection solutionThe formula of (1) is ninhydrin: vitamin C: phenol ═ 1:1:1 (v/v/v). The remaining raw materials and reagents, unless otherwise specified, are commercially available.
Example 1
In the embodiment, the FUS protein is used as a targeting receptor to design the self-assembly recognition peptide fragment beta4The self-assembly recognizes peptide fragment beta4The amino acid sequence of (A) is as shown in SEQ ID NO: 4: beta is a4(IKVSFA) and the molecular structural formula is shown in figure 3;
the synthesis steps are as follows:
(1) weighing 300mg of resin, putting the resin into a 10mL polypeptide solid phase synthesis tube, adding 7mL of Dimethylformamide (DMF), and swelling for 4 h; extracting DMF, carrying out Fmoc deprotection by using Fmoc deprotection agent, and mixing for 15min by shaking table; removing Fmoc deprotection agent, adding DMF and Dichloromethane (Dichloromethane, DCM) to wash resin for 3 times alternately, taking 10 resins from polypeptide solid phase synthesis tube, adding ninhydrin detection solution, heating to detect positive (the resin turns into dark blue), and identifying peptide segment beta according to self-assembly4The amino acid sequence of (A) is inoculated with the next amino acid to carry out amino acid condensation reaction;
(2) recognition of peptide fragment beta by self-assembly4The amino acid sequence of the polypeptide is selected, amino acid is mixed with O-benzotriazole-tetramethylurea hexafluorophosphate according to the ratio of 1:1(v/v), the mixture is dissolved by reaction liquid, and then the mixture is put into a polypeptide solid phase synthesis tube and stirred for reaction for 1 hour; taking 10 resins from a polypeptide solid phase synthesis tube, adding ninhydrin detection solution, the result is negative (no color change) to prove that the condensation reaction is successful, pumping out the excess liquid, and adding DMF and DCM to alternately wash the resin for 3 times to obtain the peptide resin condensed with the first amino acid; repeating Fmoc deprotection-amino acid condensation reaction on the peptide resin condensed with the first amino acid until the last amino acid is coupled and deprotection is completed;
(3) washing the resin with methanol for 3 times, concentrating the resin volume to 1/3 of the original volume, and continuously pumping for 15min to remove methanol; taking out the synthesized peptide resin, cracking the peptide resin in a lysate for 4 hours at 25 ℃, filtering the resin, evaporating the resin to dryness in a rotary evaporator, precipitating the polypeptide by using anhydrous ether (ice bath), and centrifuging to remove the anhydrous ether;
(4) washing the polypeptide with diethyl ether for several times, oven drying in a vacuum drying oven to obtain crude peptide product, purifying with reverse phase preparative HPLC, and purifying with HPLC purity>90 percent of the peptide is pure peptide, and the self-assembly recognition peptide fragment beta is obtained after the pure peptide is subjected to mass spectrum characterization and identification4And freeze-drying and storing at-20 ℃ for later use.
Example 2
This example compares the self-assembly recognition peptide fragment of SEQ ID NO: 4: beta is a4(IKVSFA) for functional characterization. Adopting Surface Plasma Resonance Imaging (SPRi) technology to carry out multi-self-assembly identification on peptide fragment beta4And (4) detecting the dissociation constant of the protein. The detection method comprises the following steps:
first, the gold plate was activated with 10mM disodium hydrogen phosphate/dimethyl sulfoxide solution at room temperature for 1 hour. Thereafter, the excess disodium hydrogen phosphate solution was washed off with clear water. The FUS protein (1mg/mL) solution was spotted on a gold plate, and 0.5. mu.L of the FUS protein solution was required for each spot. The spotted gold plate was placed in a wet box and allowed to stand overnight at 4 ℃. Subsequently, the excess protein solution was washed off with clear water and the plates were blocked with 5 vol% bovine serum albumin/water solution for 2h at 25 ℃. The gold plate was washed clean with clean water and the surface of the gold plate was blow dried with nitrogen. And pressing a matched cover glass, manufacturing a gold chip, and then, processing the gold chip on a machine to carry out SPRi measurement. The mobile phase used in the measurement was beta at 0.125mM, 0.25mM, 0.5mM, 1mM and 2mM in PBS buffer4The solution and the regeneration solution are 0.2M phosphoric acid solution.
The detection result is shown in figure 4, and the self-assembly recognition peptide fragment beta4Binding dissociation constant K to FUS proteinDIs 1.81X 10-5mol/L, description of beta4Can be specifically combined with FUS protein, and can be used as a targeting peptide of the FUS protein.
Example 3
In the embodiment, the polypeptide targeting drug beta is designed by taking FUS protein as a targeting receptor4-TAT, said polypeptide is targeted to drug β4-TAT amino acid sequence as shown in SEQ ID NO: 22: IKVSFAYGRKKRRQRRR, and the molecular structural formula is shown in figure 5;
synthetic method referring to example 1, only the beta4Corresponding substitution of the amino acid sequence of (A) by beta4-TAT.
Example 4
This example shows the polypeptide-targeting drug beta obtained in example 34-TAT for functional characterization. Polypeptide targeting drug beta by SPRi technology4-determination of the dissociation constant of the TAT protein.
Detection method referring to example 2, the beta4Molecular correspondence replacement by beta4TAT molecules, the mobile phase used in the measurement being 3. mu.M, 5. mu.M, 10. mu.M, 50. mu.M and 100. mu.M of beta.in PBS buffer4-TAT solution.
The detection result is shown in figure 6, and the polypeptide targeted drug beta4-binding dissociation constant K of TAT to FUS proteinDIs 2.65X 10-5mol/L indicates the polypeptide target drug beta after the modification of the cell-penetrating peptide segment4TAT can be specifically combined with FUS protein, and can be used as a targeting peptide of the FUS protein.
Example 5
In the embodiment, an escherichia coli system is used for expressing recombinant protein FUS-TagRFP-MBP-His in vitro, and the polypeptide targeted drug beta obtained in the embodiment 3 is verified4-TAT is capable of precipitating the aggregation of FUS TagRFP-MBP-His protein. The recombinant protein marks red fluorescent protein TagRFP, MBP protein (maltose binding protein) and His tag protein for FUS protein. Wherein, the TagRFP protein is used for fluorescent labeling, the MBP protein is used for improving the protein water solubility, and the His tag protein is used for purification. The experimental method is as follows:
a10. mu.M solution of FUS-TagRFP-MBP-His protein was prepared and observed under a confocal microscope, and as a result, as shown in FIG. 7, β was added4-TAT polypeptide targeting molecule to a final concentration of 100. mu.M, observed under a confocal microscope, and the results are shown in FIG. 8.
In FIG. 7, a small number of protein aggregates appeared, and in FIG. 8, a large number of micron-sized aggregates were found, indicating that the polypeptide targets drug β in vitro4TAT enables FUS-TagRFP-MBP-His protein aggregation precipitation.
Example 6
This example demonstrates polypeptide-targeted drug β on a cellular level4-TAT enables aggregation precipitation of FUS protein, SKBr-3 cell line was selected for experiments. The experimental method is as follows:
FUS-TagRFP plasmid was constructed, transfected into SkBr-3 cells using Lipofectamine 3000 reagent, to allow SkBr-3 to express FUS protein with red fluorescent protein, and FUS was visualized under a confocal microscope. To about 1051mL of 100. mu.M beta.was added to each of the FUS protein-highly expressed SKBr-3 cells4TAT polypeptide targeted drug, real-time monitoring the aggregation condition of FUS protein under a confocal microscope at normal temperature.
The experimental result is shown in fig. 9, FUS protein exists in a dispersed state in the cell nucleus at 0min, and the FUS protein gradually aggregates with the passage of time, and the FUS protein aggregates can be dispersed in the whole cell until 20 min. The results indicate that the polypeptide targets drug beta4-TAT polypeptide material is capable of recognizing binding to and co-aggregating with FUS proteins.
Example 7
This example demonstrates polypeptide-targeted drug β on a cellular level4-TAT-NBD enables aggregation precipitation of FUS protein, and SKBr-3 cell line was selected for experiments. The experimental method is as follows:
to about 1051mL of 100. mu.M beta.was added to each of the FUS protein-highly expressed SKBr-3 cells4-TAT-NBD polypeptide targeting drug at 37 ℃ with 5 vol% CO2After incubation for 4h in a cell incubator, the cells were fixed with 4 vol% paraformaldehyde for 30min, the cell permeability was increased with 0.3 vol% Triton-X-100 surfactant for 10min, the cells were incubated with FUS protein primary antibody and placed in a shaker at 4 ℃ overnight, and finally the goat anti-rabbit secondary antibody was labeled with Alexa Fluor 647 and incubated for 1h in the dark.
The results are shown in FIG. 10, where a is the immunofluorescence localization confocal image of the nucleus and b is β4Immunofluorescence co-localization confocal image of-TAT-NBD, c is an immunofluorescence co-localization confocal image of FUS protein, d is a-cThe superimposed immunofluorescence co-localization confocal image can see that the FUS protein in the cell is presented in an aggregation state and is combined with beta4the-TAT-NBD polypeptide was well co-localized, indicating that beta4-TAT-NBD is capable of aggregating FUS proteins at the cellular level.
Example 8
This example demonstrates polypeptide-targeted drug beta4-TAT-NBD and beta4-TAT inhibition efficiency on cell growth, two systems, SkBr-3 cell line and HUVEC cell line, were selected for experiments, the SkBr-3 cell line being FUS protein over-expressed and the HUVEC cell line being FUS protein under-expressed. The experimental method is as follows:
SkBr-3 cells and HUVEC cells were plated in 96-well plates at 5000 cells per well, respectively, and attached for 24 h. Then, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 60. mu.M, 80. mu.M, 90. mu.M and 100. mu.M of beta. were added, respectively4-TAT/Medium solution or beta4-TAT-NBD/media solution. And (4) incubating for 24h, adding a Cell counting kit-8 (CCK-8) and detecting the Cell survival rate.
The experimental results are shown in fig. 11 and 12, and it can be seen from fig. 11 that the polypeptide targets the drug β4-TAT has certain cytotoxicity on SKBr-3 but has no obvious cytotoxicity on HUVEC, and the result indicates that the polypeptide targets a drug beta4-TAT has strong cytotoxicity to FUS protein overexpression and has certain selectivity. As can be seen from FIG. 12, β4-TAT-NBD has obvious cytotoxicity on both cell lines, which indicates that hydrophobic small molecule NBD can enhance the toxicity of polypeptide-targeted drugs. But, in contrast, was less toxic to HUVEC cells than SKBr-3 cells.
Example 9
This example targets drug beta to a polypeptide4-TAT-NBD and beta4Mechanism of cell death by TAT was explored and validated by Western immunoblotting experiments with expression of the classical pathway LC3 protein of autophagy, using untreated SKBr-3 cells as a control, beta4-TAT (100. mu.M) or β4-TAT-NBD (100. mu.M) treated 4h of SKBr-3 cells as experimental group. The experimental method is as follows:
(1) total protein extraction
Adding 50 mu L of 100mM PMSF storage solution into 5mL of cold RIPA lysate, mixing uniformly, and placing on ice for later use;
② removing culture medium from adherent cells, washing twice with cold PBS, adding calculated cell lysate (every 10 times)71mL of lysate is required for the cells); scraping off the cells with a cell scraper on ice;
thirdly, transferring the scraped cell lysate into an Ep tube, and reversing the cell lysate for 25 min;
fourthly, after cracking, centrifuging for 30min at 4 ℃ and 12000rpm, taking supernatant and filling the supernatant into a new Ep tube.
(2) Protein quantification
Preparing a protein quantitative working solution: the ratio of the BCA reagent to the Cu reagent is 50:1, and in the experiment, 100 mu L of the Cu reagent is added into 5mL of the BCA reagent to prepare a working solution;
preparing standard solutions of 0mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 3mg/mL respectively by using a BSA standard substance for drawing a standard curve;
and thirdly, adding the standard substance and the sample into the working solution, transferring the working solution to a 96-well plate for incubation under the incubation condition of 37 ℃ for 30 min. Measuring the absorption wavelength at 562nm by using an enzyme-linked immunosorbent assay;
fourthly, calculating the protein concentration of the sample according to the standard curve, and adjusting the consistency of the protein concentration by using a diluent (PBS);
adding 5 Xsample buffer solution, and boiling for 10min with metal bath to denature protein.
(3) Protein electrophoresis
Taking out 15% of prefabricated gel, putting the prefabricated gel into an electrophoresis tank, adding 1 x electrophoresis solution, respectively holding two sides of a comb by two hands, and upwards pulling up comb teeth;
injecting 20 microliter of protein sample into the protein loading hole;
regulating the electrophoresis voltage to be 90V, setting the time to be 90min, and carrying out protein electrophoresis;
fourthly, after the electrophoresis is finished, the protein is subjected to membrane conversion by using a wet conversion method, wherein the membrane conversion condition is 300mA for 1.5 hours;
fifthly, taking out the membrane after the membrane is completely transferred, and incubating and sealing the membrane for 2 hours at normal temperature by using 5 percent of skimmed milk powder.
(4) Antigen antibody immune response
Cutting and separating target bands, adding corresponding primary antibody, and incubating for 16h at 4 ℃.
② recovering primary antibody, washing the membrane with TBST for 5 times, 7min each time.
And thirdly, incubating the secondary antibody for 2 hours at normal temperature.
Fourthly, recovering the secondary antibody, and washing the membrane for 5 times with TBST, 7min each time.
Adding ECL luminescent agent to incubate the membrane, and detecting the target strip by using an exposure instrument.
The results of the experiments are shown in FIGS. 13 and 14, where β is4-TAT and beta4the-TAT-NBD treated SkBr-3 cells produced more LC3II protein and the LC3II/LC3I values were greater, indicating that SkBr-3 cells initiate autophagy. The results indicate that the cell death mechanism is cell death by excessive autophagy.
Example 10
In the embodiment, the polypeptide targeting drug beta is designed by taking FUS protein as a targeting receptor3-TAT and β3-TAT-NBD, said polypeptide targeting drug beta3-TAT has the amino acid sequence as shown in SEQ ID NO: 23: EATVSFYGRKKRRQRRR, and the molecular structural formula is shown in figure 15;
synthetic method referring to example 1, only the beta4Corresponding substitution of the amino acid sequence of (A) by beta3-TAT and beta3-TAT-NBD.
Example 11
This example provides β obtained in example 103-TAT and beta3-TAT-NBD. Two systems, SkBr-3 cell line and HUVEC cell line, were chosen for experiments, the SkBr-3 cell line was FUS protein overexpression and the HUVEC cell line was FUS protein underexpression. The experimental method is as follows:
SkBr-3 cells and HUVEC cells were plated in 96-well plates at 5000 cells per well, respectively, and attached for 24 h. Then, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 60. mu.M, 80. mu.M, 90. mu.M and 100. mu.M of beta. were added, respectively3-TAT/Medium solution or NBD-beta3-TAT/medium solution. And (4) incubating for 24h, adding a Cell counting kit-8 (CCK-8) and detecting the Cell survival rate.
The experimental results are shown in fig. 16 and 17, and it can be seen from fig. 16 that the polypeptide targets the drug β3Toxicity of TAT on both cells vs. beta4There was a significant reduction in-TAT. Has certain cytotoxicity on SKBr-3 cells and unobvious cytotoxicity on HUVEC cells, and the result shows that the polypeptide targeted drug beta3TAT is slightly toxic to cells over-expressing FUS protein and not toxic to cells under-expressing FUS protein. As can be seen from FIG. 17, β3-TAT-NBD has obvious cytotoxicity to SkBr-3 cells, which indicates that hydrophobic small molecule NBD can enhance the toxicity of polypeptide targeted drugs. At the same time beta3-TAT-NBD has no obvious toxicity to HUVEC cells, which indicates that the molecule has certain selectivity.
Example 12
In the embodiment, the Bub-1 protein is used as a targeting receptor to design a polypeptide targeting drug beta10-TAT, said polypeptide is targeted to drug β10-TAT has the amino acid sequence as shown in SEQ ID NO: 24: FYSAHLYGRKKRRQRRR, and the molecular structural formula is shown in figure 18;
synthetic method referring to example 1, only the beta4Corresponding substitution of the amino acid sequence of (A) by beta10-TAT.
Example 13
This example provides β obtained in example 1210-results of cytotoxicity assays for TAT. U-2-OS and SH-SY5Y cell lines were selected for experiments, the U-2-OS cell line being over-expressed for the Bub-1 protein and the SH-SY5Y cell line being under-expressed for the Bub-1 protein. The experimental method is as follows:
U-2-OS and SH-SY5Y cells were plated in 96-well plates at 5000 cells per well, adherent for 24h, respectively. Then 3.9. mu.M, 7.8. mu.M, 15.6. mu.M, 31.25. mu.M, 62.5. mu.M, 125. mu.M, 250. mu.M and 500. mu.M of beta.were added, respectively10-TAT medium solution. And (4) incubating for 24h, adding a Cell counting kit-8 (CCK-8) and detecting the Cell survival rate.
The experimental result is shown in FIG. 19, and it can be seen from FIG. 19 that the polypeptide targets the drug β10-TAT has obvious cytotoxicity on U-2-OS but not on SH-SY5Y, and the results show that the polypeptide targets the drug beta10-TAT has strong cytotoxicity to the over-expression of the Bub-1 protein and has certain selectivity.
In summary, the present invention provides a polypeptide-targeted drug, which comprises a cell-penetrating peptide segment and a self-assembly recognition peptide segment. The self-assembly recognition peptide segment can recognize and aggregate with homologous protein, and induce excessive autophagy death of tumor cells. The polypeptide targeted drug is prepared by a solid-phase synthesis method, and the method is simple. The polypeptide targeted drug can be applied to the preparation of anti-tumor drugs and has important practical significance for the clinical treatment of tumors.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> national center for Nano science
<120> polypeptide targeted drug, preparation method and application thereof
<130> 2021-10-30
<160> 24
<170> PatentIn version 3.3
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Ile Phe Val Gln Gly Leu
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Arg

Claims (10)

1. The polypeptide-targeted drug is characterized by comprising a cell-penetrating peptide segment and a self-assembly recognition peptide segment for recognizing homologous proteins;
the self-assembly recognition peptide segment is a beta-sheet peptide segment in a protein structure, and the sequence of the beta-sheet peptide segment comprises any one of amino acid sequences shown as SEQ ID NO. 1-14.
2. The polypeptide-targeted drug of claim 1, wherein the cell-penetrating peptide segment is connected with the self-assembly recognition peptide segment through an amide bond;
preferably, the C end of the cell-penetrating peptide segment is connected with the N end of the self-assembly recognition peptide segment by an amido bond.
3. The polypeptide-targeted drug of claim 1 or 2, wherein the sequence of the self-assembly recognition peptide fragment comprises an amino acid sequence shown as SEQ ID NO. 4.
4. The polypeptide-targeted drug of any one of claims 1 to 3, wherein the sequence of the membrane-penetrating peptide fragment comprises any one of the amino acid sequences shown as SEQ ID NOS 15 to 21.
5. The polypeptide-targeted drug of claim 4, wherein the sequence of the cell-penetrating peptide segment comprises an amino acid sequence shown as SEQ ID NO. 15.
6. The polypeptide-targeted drug of any one of claims 1 to 5, wherein the homologous protein comprises a cytoplasmic protein or a nuclear protein;
preferably, the homologous protein comprises any one of FUS protein, Bub-1 protein, TDP-43 protein, K-Ras protein or MycFUS protein, and is preferably FUS protein.
7. The polypeptide-targeted drug of any one of claims 1 to 6, wherein the N-terminus of the polypeptide-targeted drug is linked to a hydrophobic small molecule;
preferably, the hydrophobic small molecule comprises any one of NBD, Nap, porphyrin, or benzoic acid, or a combination of at least two thereof.
8. The method for preparing a polypeptide-targeted drug according to any one of claims 1 to 7, wherein the method for preparing comprises a solid phase synthesis method.
9. Use of the polypeptide-targeted drug of any one of claims 1 to 7 for the preparation of an anti-tumor drug.
10. A method of inducing autophagy death in a cell, the method comprising: co-culturing the polypeptide-targeted drug of any one of claims 1-7 with a cell;
preferably, the concentration of the polypeptide targeting drug is 10-100 mu M, preferably 90-100 mu M;
preferably, the co-culture time is 0.5-24 h, preferably 3-5 h.
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CN114560951A (en) * 2022-03-28 2022-05-31 哈尔滨医科大学 Polypeptide-based molecule for targeting fibronectin to start assembly and application thereof
CN115845073A (en) * 2022-08-04 2023-03-28 国家纳米科学中心 Co-assembled nano material capable of realizing targeted deformation and preparation method and application thereof
CN116496410A (en) * 2023-03-23 2023-07-28 湖大粤港澳大湾区创新研究院(广州增城) Polypeptide derivative and application thereof
CN116813803A (en) * 2023-08-25 2023-09-29 北京科技大学 Polypeptide nano material for efficiently loading rapamycin as well as preparation method and application thereof
CN117229423A (en) * 2023-11-10 2023-12-15 北京科技大学 Polypeptide nano material for binding collagen and preparation method and application thereof

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CN112358531A (en) * 2020-11-09 2021-02-12 国家纳米科学中心 Polypeptide targeting HER2 protein and application thereof

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CN112358531A (en) * 2020-11-09 2021-02-12 国家纳米科学中心 Polypeptide targeting HER2 protein and application thereof

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CN114560951A (en) * 2022-03-28 2022-05-31 哈尔滨医科大学 Polypeptide-based molecule for targeting fibronectin to start assembly and application thereof
CN115845073A (en) * 2022-08-04 2023-03-28 国家纳米科学中心 Co-assembled nano material capable of realizing targeted deformation and preparation method and application thereof
CN115845073B (en) * 2022-08-04 2024-01-30 国家纳米科学中心 Co-assembled nanomaterial capable of achieving targeted deformation and preparation method and application thereof
CN116496410A (en) * 2023-03-23 2023-07-28 湖大粤港澳大湾区创新研究院(广州增城) Polypeptide derivative and application thereof
CN116496410B (en) * 2023-03-23 2023-10-10 湖大粤港澳大湾区创新研究院(广州增城) Polypeptide derivative and application thereof
CN116813803A (en) * 2023-08-25 2023-09-29 北京科技大学 Polypeptide nano material for efficiently loading rapamycin as well as preparation method and application thereof
CN116813803B (en) * 2023-08-25 2023-11-28 北京科技大学 Polypeptide nano material for efficiently loading rapamycin as well as preparation method and application thereof
CN117229423A (en) * 2023-11-10 2023-12-15 北京科技大学 Polypeptide nano material for binding collagen and preparation method and application thereof
CN117229423B (en) * 2023-11-10 2024-02-06 北京科技大学 Polypeptide nano material for binding collagen and preparation method and application thereof

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