CN104177476B - The polypeptide of a kind of targeted human cancerous cell and application thereof - Google Patents

Abstract

The present invention relates to polypeptide and the application thereof of a kind of targeted human cancerous cell.Described polypeptide is as shown in below general formula: YX₁X₂X₃X₄X₅C；The amino acid residue of described polypeptide is L-type and/or D type；The invention still further relates to the conjugate that obtained by described polypeptide and carrier conjugation and this polypeptide, its bivalent formed or multivalent for preparing treatment, prevent or diagnose the application in the medicine of cancer or imaging preparation.Many Toplink of the present invention and cancer cell marker people's Aminopeptidase N (Aminopeptidase N, APN) specific binding, compensate for the biological preparation such as antibody and prepare the shortcomings such as loaded down with trivial details, poor stability, somewhat expensive, penetration power are weak, can be used for preparing diagnostic reagent and the targeted probes of cancer.

Description

A polypeptide targeting human cancer cells and its application

technical field

The present invention relates to the field of medicinal chemistry, in particular to a polypeptide and its application, in particular to a polypeptide targeting human cancer cells, a product derived from the peptide and capable of binding to human aminopeptidase protein, the above polypeptide or its The application of the derived product in the preparation of anticancer drugs or imaging preparations.

Background technique

Cancer is a leading cause of death worldwide. Data show that in 2012 there were approximately 14.1 million new cancer cases and 8.2 million cancer deaths worldwide, compared with 12.7 million and 7.6 million in 2008. The most common cancers diagnosed worldwide were lung cancer (1.8 million, 13%), breast cancer (1.7 million, 11.9%), and colorectal cancer (1.4 million, 9.7%), and the leading cause of death was lung cancer (1.6 million, 19.4%), liver cancer (800,000, 9.1%) and gastric cancer (700,000, 8.8%).

The invasion and metastasis of cancer cells is one of the basic characteristics of malignant tumors, and it is also the main reason for the failure of clinical treatment and the death of patients. A prerequisite for cancer metastasis is the formation of new blood vessels. Therefore, at present, tumor-targeted therapy targeting neovascularization is a hot spot in the field of cancer research.

Human aminopeptidase N (Aminopeptidase N, APN) is a glycoprotein composed of 967 amino acid residues. It is expressed at a high level on the cell surface, plays an important role in the invasion and metastasis of tumor cells and the formation of tumor new blood vessels, and directly leads to and participates in the process of tumor cell infiltration and penetration of the basement membrane to metastasize.

At present, the targeted diagnostic and therapeutic drugs for APN are mainly antibody drugs. Although antibodies are widely used in clinical diagnosis due to their specific targeting, they have disadvantages such as cumbersome preparation, poor stability, high cost, and weak penetration. Therefore, in order to improve the specificity and accuracy of the diagnosis and treatment of tumor metastasis and make up for the defects of antibodies, it is urgent to seek to design small molecule probes for APN as an effective method for the detection and treatment of cancer.

Peptide drugs and diagnostic probes have the characteristics of low cost, small molecular weight, good biocompatibility, and strong penetration. The trend of antibody diagnostic reagents. In cancer research, finding highly specific peptide drugs and high affinity peptide probes against cancer cells has become a key issue. Polypeptides are composed of amino acids as the basic unit, and the diversity of amino acid arrangements determines that polypeptides vary widely in sequence, conformation, and function. Therefore, for the tumor metastasis marker APN, it is an effective way to solve the above problems by screening specific targeting polypeptides from high-throughput polypeptide libraries, and then developing them into diagnostic reagents and therapeutic drugs for cancer.

Contents of the invention

The purpose of the present invention is to provide a polypeptide and its application, especially a polypeptide targeting human cancer cells, a product derived from the peptide and capable of binding to human aminopeptidase protein, and the above polypeptide or its derivative products Application in the preparation of anticancer drugs or imaging preparations.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides a polypeptide targeting human cancer cells, the general formula of the amino acid sequence of the polypeptide is:

YX ₁ X ₂ X ₃ X ₄ X ₅ C

Wherein, Y is tyrosine; X1 is _a hydrophilic amino acid, preferably valine or glutamic _acid ; X2 is a hydrophilic amino acid, preferably valine or glutamic acid; _X3 is an aromatic amino acid , preferably tyrosine; X ₄ is a basic amino acid, preferably histidine; X ₅ is a neutral or hydrophilic amino acid, preferably leucine or serine; C is cysteine.

The polypeptide of the present invention is composed of at least 7 amino acid residues from the amino terminal of one of the sequences 1-4 in the sequence listing; preferably, the polypeptide is composed of amino acids shown in one of the sequences 1-4 in the sequence listing residue composition.

The amino acid sequences of the four polypeptides of the present invention are respectively: AP-1: YVEYHLC; AP-2: YEKYHSC; AP-3: YVENGYC; AP-4: YEVGHRC.

The polypeptides of the present invention also contain substituents which are alkoxy, alkanoyl or amide.

Preferably, in the present invention, the amino acid residues constituting the polypeptide are L-form and/or D-form.

In the second aspect, the present invention also provides a polypeptide conjugate obtained by coupling the polypeptide described in the first aspect of the present invention with a carrier; the carrier is a drug, toxin, cytokine, radioactive element, carrier protein, enzyme , lectins, fluorophores, quantum dots, or high absorptivity chromophores.

The carrier can be selected according to the specific purpose. For example, the carrier can be a drug or cytokine, which can target the drug or cytokine to cancer cells, or radioactive elements, carrier proteins, enzymes, lectins, fluorescent groups, quantum Activate the chromophore with high absorbance coefficient as the carrier.

Preferably, the carrier is an enzyme or a fluorescent group; the enzyme is preferably horseradish peroxidase; and the fluorescent group is preferably fluorescein isothiocyanate.

The polypeptide of the invention has a strong binding force to the cancer cell marker APN and has good specificity.

In the third aspect, the present invention also provides a kit comprising the polypeptide as described in the first aspect of the present invention or the polypeptide conjugate as described in the second aspect.

The kit of the invention can be used to detect human cancer cells or human aminopeptidase N protein.

In the fourth aspect, the present invention also provides a bivalent or multivalent form of the polypeptide according to the first aspect of the present invention, which has the property of targeting human aminopeptidase N protein.

Preferably, said bivalents or multivalents are formed by covalent or non-covalent linkage with multimers.

The polymer can be selected according to the specific purpose, for example, it can be polyethylene glycol (PEG) or cyclodextrin and the like.

In the fifth aspect, the present invention also provides the polypeptide described in the first aspect of the present invention or the bivalent or multivalent body described in the fourth aspect in the preparation of drugs or imaging preparations for treating, preventing or diagnosing cancer Applications.

The cancer includes but not limited to: breast cancer, lung cancer, gastric cancer, liver cancer or cervical cancer.

The polypeptide of the present invention has the effect of targeting the APN protein, and can be used as a target to increase the content of drugs or drug-loaded carriers such as nanomaterials, liposomes, etc. in APN-positive cells, and then add pharmaceutically acceptable adjuvants or adjuvants agents to make new and more effective targeted anticancer drugs.

Compared with the prior art, the present invention has at least the following beneficial effects:

The polypeptide of the present invention can target APN-positive cells and has strong selectivity, and the polypeptide involved in the present invention can be prepared by chemical synthesis, has high purity, small molecular weight, strong specificity, no immunogenicity, and is safe and reliable .

Description of drawings

Figure 1 is a schematic diagram of the chemical formula for the construction of a peptide library for screening APN-targeted polypeptides.

Fig. 2 is the surface plasmon resonance imaging (SPRi) method to detect the binding effects of AP-1, AP-2, AP-3 and AP-4 on the APN protein respectively.

Fig. 3 is the specific combination of AP-1, AP-2, AP-3 and AP-4 with APN positive cell SKOV-3 and APN negative cell 293A respectively;

Among them, Figure 3-(a)～(d) are the specific binding of AP-1, AP-2, AP-3 and AP-4 to APN positive cells SKOV-3 respectively, and Figure 3-(e) is the control polypeptide SP has no combination with APN positive cells SKOV-3;

Figure 3-(f)～(i) are the specific binding of AP-1, AP-2, AP-3 and AP-4 to APN negative cell 293A respectively, and Figure 3-(j) is the control polypeptide SP and APN negative Cell 293A did not bind.

Figure 4 shows that AP-1 and AP-2 specifically recognize the APN protein site on the surface of SKOV-3 cells.

Figure 5 shows the specific binding of AP-1, AP-2, AP-3 and AP-4 to the human liver cancer cell line HepG2, respectively.

Wherein: AP-1, AP-2, AP-3 and AP-4 respectively correspond to sequences 1-4 in the sequence listing.

detailed description

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Construction of polypeptide library and screening of APN targeting polypeptide

1. Experimental instruments and materials

N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium hexafluorophosphate (HBTU), hexahydro Pyridine, triisopropylsilane (TIS), ethanedithiol (EDT), N,N dimethylformamide (DMF), anhydrous ether, resin, methanol, various Fmoc protected amino acids, peptide synthesis tubes, shaking Bed, vacuum water pump, rotary evaporator, above reagents and materials were obtained from commercial sources.

2. Synthesis of "one bead, one object" polypeptide library

The peptide library was synthesized by the Fmoc solid-phase peptide synthesis method, and the chemical formula constructed by screening the peptide library of the APN-targeted peptide is shown in Figure 1. The specific method is to randomly couple amino acids one by one to the solid-phase resin by means of mixed splitting, and then remove the side chain protecting group under strong acid, and then carry out screening.

(1) Weigh 200 mg of Tentagel-S-NH ₂ resin, cycle according to the solid-phase peptide synthesis procedure, add 200 mg of HBTU equivalent to Met, and HBTU equivalent to Cys for two cycles of reaction. After the reaction is completed, divide the resin into 8 parts, and add 40 mg of Asn, Arg, Leu, Asp, Gly, Ser, His, Tyr and the same amount of HBTU to each tube for coupling. After the coupling is completed, Mix 8 tubes of resin, deprotect and wash. Divide the resin into 8 parts, and add 40 mg of Asn, Arg, Leu, Asp, Gly, Ser, His, Tyr and the same amount of HBTU to each tube for coupling. After the coupling is completed, put 8 tubes of resin Mix, deprotect, wash. Divide the resin into 8 parts, and add 40 mg of Asn, Arg, Leu, Asp, Gly, Ser, His, Tyr and the same amount of HBTU to each tube for coupling. After the coupling is completed, put 8 tubes of resin Mix, deprotect, wash. Then divide the resin into 4 parts, add 50 mg of Val, Glu, Ile, Lys and the same amount of HBTU to each tube for coupling, after the coupling is completed, mix the 4 tubes of resin, deprotect and wash. Then divide the resin into 4 parts, add 50 mg of Val, Glu, Ile, Lys and the same amount of HBTU to each tube for coupling, after the coupling is completed, mix the 4 tubes of resin, deprotect and wash. Divide the resin into 4 parts, and add 50 mg of Phe, Tyr, Ala, Leu and the same amount of HBTU to each tube for coupling. After the coupling is completed, mix the 4 tubes of resin and deprotect.

Add dichloromethane and trifluoroacetic acid to the above resin in sequence to remove side chain protecting groups to obtain a dry resin loaded with a peptide library for future use.

(2) Screening of polypeptides specifically binding to APN

Wash the peptide beads in the peptide library 3 times with PBS; mix and block the peptide beads with 5% skimmed milk on a gyrator (37°C, 2h); wash the peptide beads 3 times with PBS; Dilute Biotin-labeled APND protein with % skim milk at 1:1000, then mix and incubate with peptide beads on a gyrator (37°C, 2h); wash peptide beads with PBS; use excess streptavidin-labeled magnetic beads Mix and incubate with positive peptide beads on a vortexer (37°C, 1h). Use a pipette to transfer the peptide beads at the bottom of the tube to another tube. The incubated peptide library was placed in a 1.5mL centrifuge tube, and the centrifuge tube was placed on a magnetic stand. Positive peptide beads are adsorbed to the side wall of the centrifuge tube due to the influence of magnetic force, while negative peptide beads settle to the bottom of the EP tube due to gravity.

Use a pipette to transfer the peptide beads at the bottom of the EP tube to another EP tube. The peptide beads sorted by the magnetic field were picked out one by one, and the individual peptide beads were cracked with hydrogen bromide, and the corresponding sequence information was obtained by MALDI-TOF-MS (Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry) identification, and the obtained sequence included AP-1 : YVEYHLC; AP-2: YEKYHSC; AP-3: YVENGYC; AP-4: YEVGHRC. Re-synthesized according to the above sequence, identified by MALDI-TOF and purified by HPLC for subsequent experiments.

Experimental Example 1 Detection of affinity between polypeptide and human aminopeptidase N (APN) protein by surface plasmon resonance imaging (SPRi) method

Spot 1mg/mL AP-1, AP-2, AP-3, AP-4 and 1×PBS (phosphate buffer) onto the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10min , then wash with 1×PBS for 10min, and finally wash twice with deionized water, 10min each time, immerse in 1×PBS containing 5% milk, incubate overnight at 4°C, then wash with 10×PBS for 10min, 1× Wash with PBS for 10 min, and finally wash twice with deionized water for 10 min each time, blow dry with nitrogen, and install the chip on the machine (Plexera HT surface plasmon resonance imaging system).

The mobile phase was sequentially passed through 1×PBS, 2×PBS, 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL of APN purified protein, and the SPRi signal was recorded and analyzed.

As shown in Figure 2, the binding force of AP-1, AP-2, AP-3, and AP-4 to APN protein increases gradually with the increase of APN protein concentration, but 1×PBS does not increase accordingly. It shows that the four polypeptides described in this example have strong binding to APN, and can be used as APN-targeting polypeptides for related research applications.

Experimental Example 2 Interaction of AP-1, AP-2, AP-3, AP-4 with APN-positive cells SKOV-3 and APN-negative cells 293A

AP-1, AP-2, AP-3, fluorescein isothiocyanate (FITC) conjugates of AP-4 are obtained by solid-phase synthesis, AP-1 obtained in Example 1, AP-2, Continue to couple ε-aminocaproic acid to the AP-3 and AP-4 polypeptide microbeads. In a solution of pyridine/N,N dimethylformamide/dichloromethane at a ratio of 1:5:7, FITC was mixed with peptide beads and reacted overnight.

The FITC conjugates of AP-1, AP-2, AP-3 and AP-4 were obtained after trifluoroacetic acid cleavage. Polypeptide SP was coupled with FITC according to the above method, and the SP-FITC conjugate was obtained as a control. MALDI-TOF identification and HPLC purification were used for subsequent experiments.

The human ovarian cancer cell line SKOV-3 was cultured in McCoy's 5A medium containing 100mL/L fetal bovine serum, and implanted into a round glass bottom culture dish (Φ=35mm) at a cell concentration of 1×10 ⁵ /mL, 37 After culturing for 24 hours in a 5% _CO2 cell incubator at ℃, discard the culture medium and add FITC-AP-1, FITC-AP-2, FITC-AP-3, FITC-AP-4 dissolved in McCoy's 5A medium (1mg/mL, pre-cooled in ice bath); the control group was added with the same molar concentration of SP-FITC dissolved in McCoy's 5A medium (pre-cooled in ice bath); after 40min incubation in ice bath in the dark, the peptide solution was discarded and used Wash with pre-cooled PBS 3 times.

Human embryonic kidney cell line 293A cells were cultured in DMEM medium containing 100 mL/L fetal bovine serum, implanted into a round glass-bottom culture dish (Φ=35 mm) at a cell concentration of 1×10 ⁵ /mL, at 37°C, After cultivating in 5% _CO2 cell incubator for 24h, discard the culture medium, add FITC-AP-1 dissolved in DMEM medium, FITC-AP-2, FITC-AP-3, FITC-AP-4 (1mg/ mL, pre-cooled in an ice bath); the control group was added with the same molar concentration of SP-FITC dissolved in DMEM medium (pre-cooled in an ice bath); after incubation in an ice bath in the dark for 40 min, the peptide solution was discarded and washed with pre-cooled PBS 3 times.

The fluorescence distribution in the cells was detected with a laser scanning confocal microscope (Olympus FV1000-IX81, Japan). The results are shown in Figure 3, green fluorescence was observed in SKOV-3 cells added with AP-1, AP-2, AP-3, and AP-4, while the SKOV-3 cells and 263 cells of the control polypeptide SP and AP-1, No green fluorescent signal was observed in AP-2, AP-3, AP-4 263 cells.

The above results show that the recognition of FITC conjugates of AP-1, AP-2, AP-3, AP-4 and SKOV-3 is sequence-specific, and AP-1, AP-2, AP-3, AP -4 FITC conjugates specifically bind to SKOV-3 cells that highly express APN. However, the above four polypeptides did not specifically bind to APN-negative 293A cells.

2. Cell localization of AP-1, AP-2, AP-3, AP-4

In order to observe the localization of AP-1, AP-2, AP-3, and AP-4 in SKOV-3 cells, FITC-AP-1, FITC-AP-2, FITC-AP-3, FITC-AP-4 and Hoechst33342 was used for double staining experiments on SKOV-3 cells. Hoechst 33342 is a blue fluorescent dye that reveals cell nuclei.

The results are shown in Figure 3, indicating that AP-1, AP-2, AP-3, and AP-4 are bound to the cell membrane of SKOV-3 cells, which is the same as the expression site of APN.

Experimental example 3 Specific interaction between AP-1, AP-2, AP-3, AP-4 and APN

1. The human ovarian cancer cell line SKOV-3 was cultured in McCoy's 5A medium containing 100mL/L fetal bovine serum, and implanted into a round glass bottom culture dish (Φ=35mm) at a cell concentration of 1×10 ⁵ /mL, Cultivate for 24 hours at 37°C in a 5% CO ₂ cell incubator.

2. Two hours before transfection, replace the cell culture medium with serum-free McCoy's 5A medium.

3. Add 0.5 μL lipofectamine 2000 to 100 μL serum-free McCoy’s 5A medium, then add 1 μg of APNsiRNA to serum-free McCoy’s 5A medium. After standing still for 5 minutes, the siRNA solution was slowly added to the lipofectamine 2000 solution, mixed gently, and left standing at room temperature for 15 minutes.

4. Add the mixed transfection solution dropwise to a glass-bottom culture dish (Φ=35 mm), and change to McCoy's 5A medium containing 100 mL/L fetal bovine serum after 4 hours.

5. After culturing for 24 hours, discard the culture medium, add FITC-AP-1 and FITC-AP-2 (1 mg/mL) dissolved in McCoy's 5A medium, and incubate for 40 minutes in an ice bath in the dark, then discard the polypeptide solution, And washed 3 times with pre-cooled PBS.

The fluorescence distribution in the cells was detected with a laser scanning confocal microscope (Olympus FV1000-IX81, Japan), and the results are shown in FIG. 4 . Both AP-1 and AP-2 specifically combined with APN-positive SKOV-3 cells. However, RNAi SKOV-3 in cells treated with RNA interference (RNAi) did not specifically bind to AP-1 or AP-2. It has been verified that both AP-3 and AP-4 also have specific binding to APN-positive SKOV-3 cells.

Experimental example 4 Interaction between AP-1, AP-2, AP-3, AP-4 and APN positive cell HepG2

Human liver cancer cell line HepG2 cells were cultured in DMEM medium containing 100mL/L fetal bovine serum, implanted into a round glass bottom culture dish (Φ=35mm) at a cell concentration of 1×10 ⁵ /mL, 37°C, 5% After culturing in the _CO2 cell incubator for 24 h, the culture medium was discarded, and FITC-AP-1, FITC-AP-2, FITC-AP-3, FITC-AP-4 (1 mg/mL, ice bath pre-cooling); the control group was added with the same molar concentration of SP-FITC dissolved in DMEM medium (ice bath pre-cooling); after incubation for 40 min in the ice bath in the dark, the peptide solution was discarded and washed 3 times with pre-cooled PBS .

The fluorescence distribution in the cells was detected with a laser scanning confocal microscope (Olympus FV1000-IX81, Japan).

The results are shown in Figure 5, green fluorescence was observed in HepG2 cells added with AP-1, AP-2, AP-3, and AP-4, but no green fluorescence signal was observed in the HepG2 cells of the control group, indicating that AP-1, The recognition of FITC conjugates of AP-2, AP-3, AP-4 and HepG2 is sequence-specific. No green fluorescence was observed in 293A cells added with AP-1, AP-2, AP-3, and AP-4, indicating that FITC conjugates of AP-1, AP-2, AP-3, and AP-4 were associated with highly expressed APN HepG2 cells are specifically bound.

From Experimental Examples 1-4, it can be concluded that the polypeptide of the present invention has the characteristics of targeting human aminopeptidase N protein-positive tumor cells, so in practical applications, the polypeptide of the present invention can be used as a targeting polypeptide, and can kill cancer Preparations of cells are conjugated or mixed for targeted therapy of tumors.

The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (12)

1. A polypeptide targeting human cancer cells, characterized in that the polypeptide consists of the amino acid residues shown in one of the sequences 1-4 in the sequence listing. 2. The polypeptide according to claim 1, wherein the amino acid residues constituting the polypeptide are L-form and/or D-form. 3. A polypeptide conjugate, characterized in that, the polypeptide conjugate is a conjugate obtained by coupling the polypeptide according to claim 1 or 2 with a carrier; the carrier is a drug, a toxin, a cytokine , radioactive elements, carrier proteins, enzymes, lectins, fluorophores, quantum dots, or high-absorbance chromophores. 4. The polypeptide conjugate according to claim 3, wherein the carrier is an enzyme or a fluorescent group. 5. The polypeptide conjugate according to claim 4, wherein the enzyme is horseradish peroxidase; the fluorescent group is fluorescein isothiocyanate. 6. A kit, characterized in that the kit comprises the polypeptide according to claim 1 or 2 or the polypeptide conjugate according to any one of claims 3-5. 7. The kit according to claim 6, characterized in that: the kit is used to detect human cancer cells or human aminopeptidase N protein. 8. The bivalent or multivalent body formed by the polypeptide according to claim 1 or 2, characterized in that the bivalent or multivalent body has the property of targeting human aminopeptidase N protein. 9. The bivalent or multivalent body according to claim 8, characterized in that, said bivalent body or multivalent body is formed by covalent or non-covalent linkage with multimers. 10. The bivalent or multivalent body according to claim 9, characterized in that the multimer is polyethylene glycol or cyclodextrin. 11. Use of the polypeptide according to claim 1 or 2 or the bivalent or multivalent body according to any one of claims 8-10 in the preparation of medicaments or imaging preparations for diagnosing cancer. 12. The application according to claim 11, wherein the cancer is any one of breast cancer, lung cancer, gastric cancer, liver cancer or cervical cancer.