CN118384289A - Preparation method and application of tumor targeting peptide and polypeptide conjugated drug - Google Patents

Abstract

A preparation method and application of tumor targeting peptide and polypeptide coupled drug. The invention provides LYJ-1-camptothecine conjugates, and a preparation method and application thereof, belonging to the fields of polypeptide preparation and biological medicine. The method comprises the following steps: the Fmoc-based solid-phase polypeptide is adopted to synthesize LYJ-1 group, the fluorescent quenching group and camptothecine are connected to LYJ-1 through the connecting group, and then peptide cutting, separation and purification, analysis and identification and freeze drying are carried out to obtain LYJ-1-camptothecine conjugate. The LYJ-1-camptothecine conjugate has high antitumor activity, and can improve the selectivity of camptothecine to tumor cells and reduce the toxicity of camptothecine to normal cells. The invention realizes the fluorescence monitoring of the drug release in LYJ-1-camptothecine conjugate, and has good practical application value.

Description

Preparation method and application of tumor targeting peptide and polypeptide conjugated drug

Technical Field

The present invention belongs to the field of polypeptide preparation and biomedicine technology, and relates to a preparation method and application of a tumor targeting peptide and a polypeptide-coupled drug.

Background technique

Cancer, also known as malignant tumor, is caused by the uncontrolled growth and division of abnormal cells. These abnormal cells can invade surrounding tissues and organs and form distant metastases in other parts of the body. Cancer is one of the most important factors threatening human health in the world. The main methods for treating cancer are surgery, radiotherapy and chemotherapy. Small molecule drugs used for cancer chemotherapy, such as paclitaxel (PTX), camptothecin (CPT), doxorubicin (DOX), etc., have obvious tumor inhibitory effects. However, these drugs will kill normal cells indiscriminately while killing cancer cells, causing patients to experience symptoms such as nausea, vomiting, hair loss, weight loss, and decreased immunity, and have great toxic side effects. Therefore, it is urgent to develop new anticancer drugs with less toxic side effects.

CPT is an alkaloid from Camptotheca acuminata, a plant of the Vinca family, and is widely used in anti-cancer treatment in the medical field. CPT specifically binds to the topoisomerase I (Topo I)-DNA complex, preventing the topoisomerase from performing its normal functions, including unwinding the DNA superhelical structure and participating in DNA replication, recombination, repair and transcription. Therefore, when topoisomerase is inhibited by CPT, the DNA replication and repair process will be blocked, leading to cell death. However, CPT has poor water solubility, is not easy to pass through the cell membrane, and is highly toxic to normal cells, which limits the application of CPT.

SORT1 receptor, also known as neurotensin receptor 3, is a membrane-bound receptor. The functional regulation of SORT1 receptor is related to a variety of physiological and pathological processes. In recent years, SORT1 receptor has attracted widespread attention in cancer research. Studies have shown that SORT1 is highly expressed in a variety of tumor cells such as breast cancer, lung cancer, and glioma. Some studies have found that SORT1 receptor may play an important role in the proliferation, invasion, and metastasis of tumor cells. Therefore, SORT1 receptor is considered to be a potential therapeutic target and may provide new strategies and methods for cancer treatment. Peptide LYJ-1, also known as TH19P01 (amino acid sequence: Ac-GVRAKAGVRN(Nle)FKSESY-NH ₂ ), can specifically target SORT1 receptor. Compared with antibodies, peptide LYJ-1 overcomes the problem of heterogeneity; it has a small molecular weight and is easy to pass through the biological membrane barrier; it is easy to chemically synthesize and has low cost.

Summary of the invention

The main purpose of the present invention is to provide a preparation method and application of a tumor targeting peptide and a polypeptide-coupled drug.

Based on the above purpose, the technical solutions involved in the present invention are as follows:

1) The present invention provides a tumor targeting peptide and a polypeptide conjugate thereof, wherein the tumor targeting peptide and the polypeptide conjugate comprise the following amino acid residue sequence:

LYJ-1:Ac-GVRAKAGVRN(Nle)FKSESY-NH ₂

LYJ-2:Ac-GVRAK(-CPT)AGVRN(Nle)FK(-CPT)SESY-NH ₂

LYJ-3:Ac-GVRAK[-K(Dnp)-CPT]AGVRN(Nle)FK[-K(Dnp)-CPT]SESY-NH ₂

LYJ-4:Ac-GVRAK(-7-DCCA)AGVRN(Nle)FK(-Ac)SESY-NH ₂

2) The present invention provides a method for preparing the above-mentioned polypeptide and polypeptide-coupled drug, the preparation method comprising synthesizing the polypeptide by solid phase polypeptide synthesis, and connecting the polypeptide with a linking group and a small molecule drug by condensation reaction.

3) The present invention provides an evaluation of the anti-cancer or anti-tumor effect of the above-mentioned polypeptide-coupled drug.

4) The present invention provides the synthesis and application of the above-mentioned polypeptide-coupled drug release monitoring probe. LYJ-3 of the present invention is a combination of CPT and Dnp, which emits specific blue fluorescence after CPT is released, thereby realizing the monitoring of CPT release.

The present invention has the following advantages:

1) It enhances the selectivity of CPT for tumor cells and reduces the killing effect of CPT on normal cells, thereby reducing the toxic side effects of CPT.

2) Increased the uptake of CPT by tumor cells.

3) The release of the fluorescent small molecule anti-tumor drug CPT is monitored through intramolecular self-quenching. When Dnp is used in combination with CPT, the fluorescence disappears, and when CPT is released, blue fluorescence is emitted, thus monitoring the release of CPT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the solid phase polypeptide synthesis method of the present invention;

FIG2 is the chemical structural formula and primary amino acid sequence of LYJ-1 of the present invention;

FIG3 is an analytical reverse phase high performance liquid chromatogram and ESI-MS mass spectrum of LYJ-1 of the present invention;

FIG4 is the chemical structural formula and primary amino acid sequence of LYJ-2 of the present invention;

FIG5 is an analytical reverse phase high performance liquid chromatogram and ESI-MS mass spectrum of LYJ-2 of the present invention;

FIG6 is the chemical structural formula and primary amino acid sequence of LYJ-3 of the present invention;

FIG7 is an analytical reverse phase high performance liquid chromatogram and ESI-MS mass spectrum of LYJ-3 of the present invention;

FIG8 is the chemical structural formula and primary amino acid sequence of LYJ-4 of the present invention;

FIG9 is an analytical reverse phase high performance liquid chromatogram and ESI-MS mass spectrum of LYJ-4 of the present invention;

FIG10 is a cell-level evaluation of the cytotoxicity of LYJ-2 on cancer cells MDA-MB-231, 4T1 and normal cells 293T according to the present invention;

FIG11 is a cell-level evaluation of the effect of LYJ-2 on inhibiting the migration of MDA-MB-231 and 4T1 cells in the present invention;

FIG12 is a study of LYJ-2 uptake evaluated at the cellular level of the present invention;

FIG13 is a hemolytic activity test of the synthetic compounds of the present invention;

FIG. 14 shows the release of CPT monitored by the intramolecular self-quenching principle of the present invention.

Detailed ways

Implementation Examples

1. Synthesis of peptides

In this example, all target peptides were prepared using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis technology (Fmoc-SPPS). RinkAmideAm resin (substitution degree 0.27 mmol/g) was used to synthesize peptides containing amide termini.

The scale of peptide synthesis was 0.1 mmol, and the basic process of peptide synthesis is shown in Figure 1.

Weigh 500 mg of RinkAmideAM resin (1 equivalent), wash with DMF and DCM alternately (DMF 2 times/DCM 2 times/DMF 1 time/DCM 1 time/DMF 3 times, standard wash), soak the resin at room temperature for 1 to 2 hours, and then soak the resin with DMF/DCM mixed solution (4:1, volume ratio, v:v) after standard washing again, and shake in a constant temperature oscillator at 28°C for 0.5 to 1 hour to activate the resin. Then use 20% piperidine (piperidine:DMF, 1:4, v:v) to remove the Fmoc protecting group, remove twice, the first time for 6 minutes, and the second time for 12 minutes. Each amino acid is condensed twice, 40 minutes and 50 minutes respectively, and the ratio of the reactants is Fmoc-amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents. After the last amino acid is condensed, its Fmoc protecting group is removed, acetylated or connected to other small molecules, and the ratio of the acetylated reactants is acetic anhydride: DIPEA: DMF = 1:1:8 (volume ratio, v:v:v). Then, wash once with standard method and then rinse three times with DCM.

The above-synthesized targeting peptide is subjected to condensation reaction with the linking group through an amide bond, and then the linking group is condensed with the small molecule drug group through an amide bond. The reactant ratio of LYJ-2 and LYJ-3 is CPT: HATU: HOAT: DIEA = 2 times equivalent: 1.8 times equivalent: 2 times equivalent: 4 times equivalent, and the reactant ratio of LYJ-4 is coumarin: HATU: HOAT: DIPEA = 3 times equivalent: 2.8 times equivalent: 3 times equivalent: 6 times equivalent. In particular, the lysine (K) used in LYJ-3 is connected to a Dnp group, which absorbs the fluorescence of CPT, and the fluorescence of CPT can be detected after CPT is released.

The resin washed three times with DCM was evacuated with a water pump for 5 minutes, and then with an oil pump for 4 minutes, and a pre-cooled peptide cleavage reagent was added thereto, the ratio of which was TFA: phenol: water: TIPS = 88: 5: 5: 2 (v: v: v: v). It was placed in a shaker at 28 ° C for 2.5 to 3 hours, after which the reaction solution was blown into a three-necked flask with an ear bulb, the resin was washed twice with 0.5 mL of TFA, and also blown into a three-necked flask, and the reaction product was blown to 3 mL with high-purity nitrogen, and pre-cooled anhydrous ether was added to the concentrate to precipitate the target polypeptide. Centrifugation was performed to obtain a crude peptide, and anhydrous ether was added for washing and centrifugation, which was repeated three times. The product was placed in a fume hood for evaporation and drying. Then, a mixed solution of acetonitrile and water containing 1‰ TFA was used to dissolve the crude peptide product, and then the obtained crude peptide was analyzed and identified using analytical reverse HPLC and ESI-MS. The dissolved crude peptide was frozen at -80 ° C. It was freeze-dried to obtain a flocculent crude peptide. The crude peptide was dissolved again in a mixed solution of acetonitrile and water containing 1‰ TFA, separated and purified using semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), the collected pure peptide solution was frozen at -80°C, and then freeze-dried in a vacuum freeze dryer to obtain the target pure peptide.

The chemical structural formulas, primary amino acid sequences, analytical reversed-phase high performance liquid chromatography and ESI-MS mass spectra of the four targeting peptides and polypeptide conjugates prepared in the present invention are shown in FIGS. 2 to 9 .

2. Application in anti-tumor

Cytotoxicity assay

The toxicity of the targeting peptide LYJ-1 and its peptide conjugates to adherent cells was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)tetrazolium, Mecklin) method. Adherent cells MDA-MB-231 were seeded on 96-well plates at a density of 3000 cells/well and incubated for 24 hours. CPT and LYJ-2 were added to 96-well plates at 0.001, 0.01, 0.1, 1, 10, and 100 μM, respectively, and incubated for 72 hours. For the group with LYJ-1 added in advance, LYJ-1 was added 1 hour in advance for incubation, and then LYJ-2 and LYJ-1 were added in equal proportions according to the same concentration gradient as above. Then 15 μL of MTT working solution with a concentration of 5 mg/mL was added to the 96-well plate and incubated for 4 hours. The supernatant in the 96-well plate was aspirated with a syringe, and then 150 μL of DMSO was added, and the culture was continued in a CO ₂ incubator for 1 hour. Finally, the 96-well plate was shaken evenly, and the OD value was measured at 492 nm using an enzyme reader.

Adherent cells 293T or 4T1 were seeded on a 96-well plate at a density of 5000 cells/well and incubated for 24 hours. After that, complete culture medium containing 0.032, 0.16, 0.8, 4, 20, and 100 μM LYJ-2 or CPT was added to the 96-well plate and incubated for 72 hours. Then 15 μL of MTT working solution with a concentration of 5 mg/mL was added to the 96-well plate, and the cells were incubated for 4 hours. Subsequently, the supernatant in the 96-well plate was aspirated with a syringe, and then 150 μL DMSO was added, and the culture was continued in a CO ₂ incubator for 1 hour. Finally, the 96-well plate was shaken evenly and the OD value was measured at 492 nm with an enzyme reader.

As shown in Figure 10, LYJ-1 did not show obvious cytotoxic effects on these three cells. For normal cells 293T, the half-inhibitory concentration value of CPT was <0.001μM, and the half-inhibitory concentration value of LYJ-2 was 11.08μM, and the toxicity to normal cells was significantly reduced. For 4T1 cells with low expression of SORT1 receptor, the half-inhibitory concentration value of CPT was 0.03μM, and the half-inhibitory concentration value of LYJ-2 was 2.0μM. Compared with CPT, the killing effect of LYJ-2 on tumor cells was reduced by 66.7 times. However, for MDA-MB-231 cells with high expression of SORT1 receptor, the half-inhibitory concentration value of CPT was 0.6μM, and the half-inhibitory concentration value of LYJ-2 was 1.5μM; compared with CPT, the killing effect of LYJ-2 on tumor cells was reduced by 2.5 times, which was much less than the 66.7-fold reduction for 4T1 cells. In addition, for LYJ-2, the half-inhibitory concentration of LYJ-2 on high-expressing cells MDA-MB-231 increased from 1.5μM to 7.6μM by incubating the targeting peptide LYJ-1 in advance, and the killing effect on tumor cells was reduced by 5 times. The above results show that targeting peptides can improve the selectivity of CPT for tumor cells and reduce the killing effect on normal cells.

Scratch test

The scratch test was used to explore the effect of LYJ-2 on inhibiting the migration of MDA-MB-231 and 4T1 cells. The cells were placed in a 12-well plate (1×10 ⁶ cells/well) and cultured for 48 hours. Then, a sterile p1000 pipette tip was used to scratch the bottom of the well plate, with two scratches per well. The cells were rinsed three times with PBS, the scratched cells were removed, and a liquid with a concentration of 1.5 μM was prepared with fresh incomplete culture medium and added to the 12-well plate for 2 hours. It was aspirated and washed with complete culture medium, and then complete culture medium was added to continue culture for 24 hours. Photos were taken with a confocal microscope at 0 hours and 24 hours. At the same time, DMSO was used as the control group, and LYJ-2, CPT, LYJ-1, and LYJ-1+CPT were all experimental groups.

Calculation formula: Migration inhibition rate = [(0h-24h)/24h] × 100%.

The results are shown in Figure 11. After 24 hours, it can be seen that LYJ-2 can significantly inhibit the migration of MDA-MB-231 cells in high-expressing tumor cells. Compared with the LYJ-2-treated group, the inhibition degree of the CPT-treated group and the LYJ-1 and CPT non-covalently linked treatment group was significantly lower than that of LYJ-2. The above results show that the covalent linking of LYJ-1 and CPT improves the inhibitory effect on cell migration.

In low-expressing tumor cells, LYJ-2 has a certain inhibitory effect on 4T1 cell migration, but the inhibitory effect of LYJ-2 on SORT1 receptor high-expressing cells is significantly higher than that on low-expressing cells. The above results further prove that the covalent connection of targeting peptide LYJ-1 and CPT shows a stronger inhibitory ability on SORT1 high-expressing cells.

Cellular uptake experiments

In order to explore the endocytosis of LYJ-1 targeting peptide conjugates by cells, MDA-MB-231 cells were seeded in 24-well plates at 30,000 cells/well. After incubation for 24 hours, the complete medium was aspirated, washed three times with PBS, and treated with SYBRGreen I fluorescent dye for 30 minutes to fluorescently label the cell nuclei. Washed three times with PBS, fresh incomplete medium containing 10 μM LYJ-2, LYJ-4, LYJ-1+CPT, and LYJ-1+7-DCCA was added to the well plate and incubated. Under the same conditions, the cells were imaged under a confocal microscope at time intervals of 2 hours, 4 hours, 6 hours, and 12 hours.

As shown in Figure 12, as time goes by, the fluorescence intensity of CPT detected in the LYJ-2 group gradually increases. And compared with free CPT, LYJ-2 shows significantly enhanced fluorescence. The fluorescence intensity of cells treated with LYJ-1 and CPT non-covalently linked did not increase significantly, indicating that the covalent linking of the targeting peptide to CPT improves the cellular uptake of CPT.

As a control, the fluorescence intensity of coumarin was detected to increase gradually in cells treated with LYJ-4 over time. However, for the non-covalent combination of targeting peptide LYJ-1 and coumarin, the fluorescence intensity did not increase significantly over time, indicating that the covalent connection of targeting peptide and coumarin increased the cellular uptake of coumarin. The above results indicate that covalent connection is necessary for the cellular uptake of CPT and coumarin.

Hemolysis test

The hemolytic activity of LYJ-1, LYJ-2, CPT, and LYJ-1+CPT was determined using mouse red blood cells. LYJ-1, LYJ-2, CPT, and LYJ-1+CPT were weighed and prepared into solutions with concentrations between 0.03μM and 30μM. Fresh mouse blood was taken and centrifuged at a speed of 1000r/min for 5min. The supernatant was discarded, and the red blood cells in the lower layer were washed three times with PBS (centrifuged at a centrifugal speed of 1000r/min for 5min). A 5% v/v red blood cell suspension was prepared, and an equal volume of red blood cell suspension was added to the polypeptide sample solution after mixing. The concentration of the red blood cell suspension was diluted by one time and incubated in a 37°C incubator for 4 hours. The centrifuge tube was centrifuged, the supernatant was taken into a 96-well plate, and the absorbance of the mixture solution was measured at 450nm using an enzyme marker. At the same time, water was used as a positive control and PBS was used as a negative control to set up the experiment.

The hemolysis percentage was calculated according to the following formula: hemolysis percentage = [(sample-negative)/(positive-negative)] × 100%.

Figure 13 shows that within the concentration range of 0.03 μM to 30 μM, the CPT+LYJ-1 blend exhibits significantly increased hemolytic activity, while the hemolytic activity of CPT, LYJ-1 targeting peptide, and drug-peptide conjugate LYJ-2 is relatively low. It can be seen that by covalently linking CPT to the targeting peptide, the toxic side effects of CPT on red blood cells are not significantly increased.

Real-time monitoring of CPT release using the principle of intramolecular self-quenching

Weigh appropriate amounts of LYJ-3, Dnp and CPT, and prepare LYJ-3, Dnp+CPT and CPT solutions containing 5% serum and a concentration of 50 μM, respectively. Incubate in a constant temperature water bath at 37°C for 72 hours, and take samples at 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, and 72h. Take 80 μL of the mixed solution, add 20 μL of acetonitrile containing 0.1% TFA, mix well, let stand on ice for 2 minutes, then add 60 μL of acetonitrile and 400 μL of deionized water containing 0.1% TFA, shake three times, and store at -80°C. The release of CPT was monitored in real time by a fluorescence spectrophotometer.

The release of CPT can be monitored by using the principle of intramolecular self-quenching. When CPT is not released, Dnp is covalently linked to CPT, and Dnp absorbs the fluorescence of CPT. When CPT is released, since the distance between Dnp and CPT becomes farther and Dnp cannot absorb the fluorescence of CPT, the fluorescence of CPT can be detected using a fluorescence spectrophotometer. Figure 14A shows the changes in fluorescence intensity of each group over time. It can be seen that with the change of time, the fluorescence intensity of LYJ-3 continues to increase, while the fluorescence intensity of the Dnp+CPT group and the CPT group has almost no change. Figure 14B is the ratio of the fluorescence intensity of each group over time to the fluorescence intensity at zero time, showing that the fluorescence intensity of the Dnp+CPT group and the CPT group has not increased compared with the zero time. The fluorescence intensity of the LYJ-3 group gradually increases with the extension of time, indicating that the CPT in LYJ-3 is continuously released over time. The above results show that the introduction of Dnp can be used to monitor the release of CPT, and the ability of the compound to continuously release CPT within 6 days (144 hours) was observed.

The present invention improves the selectivity of CPT for tumor cells and increases the cellular uptake of CPT. The present invention covalently links Dnp and CPT to obtain LYJ-3, thereby realizing real-time fluorescence monitoring of CPT release. The present invention covalently links coumarin and LYJ-1 to obtain LYJ-4, thereby enabling real-time monitoring of the targeting peptide at the molecular and cellular levels.

Claims (5)

1. A LYJ-1-camptothecin conjugate, characterized in that the LYJ-1-camptothecin conjugate comprises the following amino acid sequence LYJ-2:Ac-GVRAK(-CPT)AGVRN(Nle)FK(-CPT)SESY-NH ₂ LYJ-3:Ac-GVRAK[-K(Dnp)-CPT]AGVRN(Nle)FK[-K(Dnp)-CPT]SESY-NH ₂ LYJ-4:Ac-GVRAK(-7-DCCA)AGVRN(Nle)FK(-Ac)SESY-NH ₂ . 2. The preparation method of the LYJ-1-camptothecin conjugate according to claim 1, characterized in that the preparation method comprises preparing the polypeptide by solid phase polypeptide synthesis, and connecting the polypeptide group with the linking group and the small molecule drug group by condensation reaction; Preferably, the polypeptide synthesis method is a solid phase polypeptide synthesis method based on 9-fluorenylmethoxycarbonyl, namely Fmoc-SPPS; preferably, the preparation method of the LYJ-1-camptothecin conjugate is as follows: The polypeptide was prepared by solid-phase condensation resin and 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis technology, and then the synthesized polypeptide group was connected with the linking group and the small molecule drug through an amide bond reaction. A peptide cleavage mixed reagent was added to the obtained condensation product, and the condensation product was cut off from the resin. The product after peptide cleavage was separated, purified, analyzed, identified and freeze-dried to obtain a LYJ-1-camptothecin conjugate. 3. Use of the LYJ-1-camptothecin conjugate according to claim 2 in the preparation of anticancer or antitumor drugs; Preferably, the tumor cells are selected from mouse breast cancer and human breast cancer; Preferably, the normal cells are selected from human kidney epithelial cell lines. 4. A pharmaceutical composition, characterized in that the composition comprises the LYJ-1-camptothecin conjugate according to claim 1. 5. Use of the LYJ-1-camptothecin conjugate according to claim 1 as a probe for monitoring drug release.