CN105949282B - FAP-targeted anti-angiogenesis peptide Z-GP-V2 and application thereof - Google Patents
FAP-targeted anti-angiogenesis peptide Z-GP-V2 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of tumor treatment, and particularly relates to FAP-targeted synthetic peptide Z-GP-V2 with an anti-tumor angiogenesis effect. The molecular weight of the synthesized peptide is 1149.6, and the sequence is as follows: Z-GPAANLLMAAS; the Fmoc solid-phase peptide is prepared by adopting an Fmoc solid-phase polypeptide synthesis method; can be used for preparing anti-tumor angiogenesis preparation. The invention obtains the new synthetic peptide Z-GP-V2 by modifying the V2 peptide by utilizing the specific substrate dipeptide Z-GP, and the new synthetic peptide shows better application effects on the aspects of improving the targeting property of the V2 peptide, improving the anti-tumor angiogenesis function of the V2 peptide, reducing the toxic and side effects of the V2 peptide and the like, and simultaneously provides new reference for a new anti-tumor angiogenesis treatment method.
Description
Technical Field
The invention belongs to the technical field of tumor treatment, and particularly relates to FAP-targeted synthetic peptide Z-GP-V2 with an anti-tumor angiogenesis effect.
Background
In recent years, the incidence and mortality of malignant tumors have increased year by year, and the malignant tumors become the leading cause of adult death in developed countries and parts of developing countries, are one of the biggest public problems in the world, and seriously threaten the health and life safety of people. Therefore, the treatment of cancer is generally concerned and highly emphasized by the international society, and the search for safe and effective anti-tumor drugs with small adverse reactions is always a focus of biomedical research. The traditional clinical treatment medicament principle is to inhibit the growth of tumor by directly killing tumor cells, however, the medicaments usually have cytotoxicity to normal cells, and when the medicaments kill the tumor cells, the medicaments are easy to cause side effects such as bone marrow suppression, low immune function, irregular bleeding, nausea and vomiting and the like to the human body, and the tumor cells are easy to generate resistance to the medicaments along with the prolonging of the use time of the medicaments. Therefore, the search for anti-tumor drugs with good effect, low toxicity and strong specificity from different approaches is the main target of the current tumor treatment, and more researchers take various stromal cells in the tumor microenvironment as targets to research the cancer treatment, and obtain a certain treatment effect, and the anti-tumor drugs have become a new hotspot of the tumor treatment research.
Tumor vascular blocking therapy, which takes tumor vascular endothelial cells as targets and inhibits tumor growth by inhibiting tumor angiogenesis, is widely regarded by people. Theoretically, tumors are blood vessel dependent malignant cells, and the growth, metastasis, recurrence and prevention of which are closely related to tumor angiogenesis. In fact, 90% of cancer patients die from vascular access metastases, which are also the greatest harm caused by tumor vessels. Therefore, the aim of treating the tumor can be fundamentally achieved only by inhibiting the growth of the tumor new blood vessels. Most of the existing endogenous angiogenesis inhibitors are fragments of some natural components in the processes of ECM and blood coagulation, but the fragments cannot effectively penetrate tissues due to large structures, and simultaneously have the defects of easy removal in a human body and high medicament using amount and application cost, thereby seriously limiting the application of the endogenous angiogenesis inhibitors in clinic. Therefore, many researchers are now working on small molecule polypeptide drugs that have similar effects to larger endogenous proteins or polypeptides.
Fibroblast Activation Protein (FAP) is a type ii serine peptidase, a member of the serine protease family, has dipeptidylpeptidase and collagenase activities, promotes tumor infiltration and metastasis and microvascular formation, is specifically expressed in the membrane and cytoplasm of stromal fibroblasts of more than 90% of epithelial tumors, including colon, breast, ovarian, bladder, and lung cancers (primary and metastatic), and positive cells are near the endothelial cells of tumor capillaries and surround tumor nodules, but are not normally expressed in normal adult tissues, benign and precancerous epithelial damaged tissues. The protease activity of FAP can enhance the invasiveness of tumor cells to extracellular matrix and promote the growth and proliferation of tumors. Research shows that FAP plays a promoting role in the whole process of tumor formation, can effectively enhance the formation of tumor new vessels, and can prevent the nutrition supply of tumor stroma source and blood vessel source by the targeted inhibition of FAP. Due to the important influence on the growth of the tumor and the characteristic that the FAP is specifically expressed in the tumor stromal fibroblasts, the FAP has important significance in the targeted therapy of the tumor.
Disclosure of Invention
Based on a dipeptide substrate (Z-GP) based on FAP and the existing Tie2 targeting peptide structure, the invention designs and provides a novel synthetic peptide substance: Z-GP-V2, thereby providing a new reference and reference for the prevention and treatment of tumors.
The detailed technical scheme adopted by the invention is as follows.
An anti-angiogenesis synthetic peptide Z-GP-V2 targeting FAP is constructed by connecting two functional domains through an alanine flexible linker, and is specifically formed by connecting a dipeptide substrate (Z-GP, N-benzyloxycarbonyl glycine proline) targeting FAP and a polypeptide fragment (V2 peptide, NLLMAAS) with anti-tumor angiogenesis targeting Tie2 through the alanine flexible linker;
the molecular weight of the peptide is: 1149.6 Da, the sequence is shown in SEQ ID NO.1, and the sequence specifically comprises: Z-Gly-Pro-Ala-Ala-Asn-Leu-Leu-Met-Ala-Ala-Ser; namely Z-GPAANLLMAAS or Z-GP-AA-NLLMAAS.
The FAP-targeted anti-angiogenesis synthetic peptide Z-GP-V2 is prepared by adopting an Fmoc solid-phase polypeptide synthesis method, and the specific process comprises the following steps:
(1) selecting Wang resin to be connected with the first Fmoc-amino acid carboxyl at the C end of the peptide to be synthesized in a covalent bond mode, taking the N end of the amino acid as the starting point of the polypeptide synthesis, and enabling the Wang resin and the carboxyl end of the next amino acid to generate dehydration condensation reaction to form a peptide bond;
(2) then, protecting groups of Fmoc-amino acid at the N end are deprotected, then the N end of the second amino acid reacts with the carboxyl of the following amino acid, and the process is repeated until the polypeptide synthesis is finished;
(3) finally, the synthesized polypeptide is cut off from the resin, and crude peptide is obtained through ether precipitation and washing;
(4) desalting, RP-HPLC analyzing and purifying to obtain the FAP-targeted synthetic peptide Z-GP-V2 for resisting tumor angiogenesis, wherein the synthetic peptide Z-GP-V2 can be frozen at-20 ℃ for later use.
The synthetic peptide Z-GP-V2 targeting FAP is used for resisting tumor angiogenesis.
It has been shown that FAP has specific endopeptidase activity and can selectively hydrolyze N-terminally blocked glycylproline dipeptide sequences, such as substrates carrying Z-Gly-Pro (Z-GP) dipeptides, and thus targeted drugs can be designed against the substrates to block the action of FAP. The research on the V2 peptide shows that the peptide is a small molecular polypeptide which acts on a tumor angiogenesis related factor receptor Tie2 specifically, and the peptide can block the combination of Ang (angiogenin) which is more critical in the process of forming blood vessels and the receptor thereof specifically, thereby blocking the angiogenesis. However, no matter the treatment mode designed for Z-GP or the application of V2 peptide, the treatment method of single targeting vascular endothelial cell receptor has certain limitation and defect, and the main reason is that the formation of new blood vessels occurs not only at the periphery of tumor cells, but also under the conditions of wound healing, endometrial periodic change, myocardial infarction, diabetes and the like, and when the normal function of VEGF is inhibited, the drugs applied to the tumor treatment can cause the occurrence of side reactions such as hypertension, proteinuria, hemorrhage, thrombotic events, gastrointestinal perforation, wound healing syndrome and the like, thereby limiting the treatment effect of the single targeting drugs to a certain extent, and influencing the popularization and application of the treatment method.
On the basis of the action of the existing single targeting drug, the Z-GP is connected with the V2 peptide with the targeting Tie2 and the anti-tumor angiogenesis activity through the alanine flexible linker, so that the novel double-targeting synthetic peptide Z-GP-V2 is obtained. The anti-angiogenesis effect of the synthetic peptide is further verified through an in vitro cell scratch experiment and a migration experiment. The verification of a mouse S180 transplanted tumor model experiment with abundant blood vessels shows that the peptide has obvious tumor inhibition effect, has an anti-tumor angiogenesis effect, has no obvious toxic or side effect, has a good medical application prospect, and provides a new idea for targeted anti-tumor vascular therapy.
In general, compared with a treatment method directly taking tumor tissues as targets, the treatment method of targeting anti-tumor angiogenesis has the advantages of difficult generation of drug resistance, strong specificity, small toxic and side effects and the like; meanwhile, one vascular endothelial cell can support the growth of 50-100 tumor cells, so that the tumor body inhibition effect can be multiplied after the tumor blood vessels are blocked. Based on the great advantages of the anti-tumor angiogenesis treatment method, the novel synthetic peptide Z-GP-V2 is obtained by modifying the V2 peptide by utilizing the specific substrate dipeptide Z-GP, and the novel synthetic peptide shows good application effects on the aspects of improving the targeting property of the V2 peptide, improving the anti-tumor angiogenesis function of the V2 peptide, reducing the toxic and side effects of the V2 peptide and the like, and simultaneously provides a new reference for the novel anti-tumor angiogenesis treatment method.
Drawings
FIG. 1 shows ESI-MS mass spectrometry identification of the prepared synthetic peptide Z-GP-V2;
FIG. 2 is a graph of the effect of synthetic peptide Z-GP-V2 on HUVECs migration in cell scratch experiments at 5 μ M;
FIG. 3 is a graph showing the effect of synthetic peptide Z-GP-V2 on the migration of HUVECs in cell migration experiments;
FIG. 4 is a graph showing the effect of synthetic peptide Z-GP-V2 on changes in body weight of S180 loaded BABL/c mice;
FIG. 5 is a graph showing the effect of synthetic peptide Z-GP-V2 on the change in the volume of transplanted tumors in S180 bearing BABL/c mice;
FIG. 6 is a graph of the effect of synthetic peptide Z-GP-V2 on the graft tumor weight of S180 bearing BABL/c mice.
Detailed Description
The technical solution of the present invention will be described in detail with reference to the following specific embodiments, but before describing the specific embodiments, the conditions of some experimental reagents and experimental equipments used in the present invention will be briefly described as follows.
Biological material:
s180 cells (murine sarcomas cells), HUVECs (human umbilical vein endothelial cells), purchased from atcc (american type Culture collection);
BALB/c female mice (50 mice) with SPF grade of 5-8 weeks are purchased from the center of experimental animals in Henan province;
experimental reagent:
wang resin synthesized by Fmoc polypeptide solid phase and L-shaped amino acid protected by Fmoc are products of Jier Biochemical (Shanghai) Co.Ltd;
washing reagents in Fmoc polypeptide solid phase synthesis, 1-hydroxybenzotriazole (HOBt), N-Diisopropylcarbodiimide (DIC), 4-Dimethylaminopyridine (DMAP), N-Dimethylformamide (DMF), methanol (MeOH), Dichloromethane (DCM), ninhydrin, piperidine, acetic anhydride, trifluoroacetic acid (TFA), acetonitrile, etc., from Tianjin Kangship Chemicals, Inc.;
RPMI-1640 medium, a product of Solambio, Beijing;
phosphate Buffered Saline (PBS) was prepared with related reagents, purchased from Mimi European chemical reagents, Inc., Tianjin;
FBS (fetal bovine serum) purchased from hangzhou sijiqing biology ltd;
experimental equipment:
a polypeptide synthesizer, south Xingshi glass instrument factory of Jiangsu Tongzhou city;
rotary evaporator RE-52, Shanghai Yangrong Biochemical apparatus works;
RP-HPLC analyzer, Shimadzu corporation, Japan;
inverted microscope AE2000, Olympus corporation.
Example 1
The synthetic peptide Z-GP-V2 provided by the invention has the molecular weight as follows: 1149.6, specifically: the peptide Z-Gly-Pro-Ala-Ala-Asn-Leu-Leu-Met-Ala-Ala-Ser is prepared by adopting an Fmoc solid-phase polypeptide synthesis method, and the specific preparation method is as follows by taking 0.2073 g of synthetic peptide Z-GP-V2 synthesized in a certain time as an example.
(1) Weighing 0.3g of Wang resin, putting the weighed resin into a polypeptide synthesizer rinsed by DMF, adding 4 ml of DMF, standing for 30min to fully swell the resin, and then pumping out the DMF by a vacuum pump.
(2) Addition of the first amino acid: weighing corresponding amounts of serine, HoBt and DIC according to a formula 1, weighing corresponding amounts of DMAP according to a formula 2, wherein the corresponding amounts are 287.55mg, 101.3475mg, 94.65 muL and 9.162mg respectively, dissolving serine and HoBt by using 4 mL of DMF, adding the serine and HoBt into a synthesizer, directly adding DIC into the synthesizer, stirring and reacting for 10min at 25-28 ℃, adding DMAP dissolved by using 1mL of DMF into the synthesizer, and continuously stirring and reacting for 2.5h at the temperature;
the formula 1 is: mass of amino acid = relative molecular mass of the amino acid × 2.5 (equivalents) × mass of resin;
the formula 2 is: mass of DMAP = relative molecular mass of DMAP x 0.25 (equivalents) × mass of resin.
(3) Washing the resin reacted in the step (2) according to the following sequence and times, namely, two times of DMF → three times of MeOH → three times of DCM → two times of DMF, washing in a shaking table in a shaking way for two minutes each time, and draining the liquid by using a vacuum pump when the washing is finished;
measuring absorbance OD of the resin and the first amino acid at a wavelength of 290nm by using a spectrophotometer, calculating a substitution value according to a formula, sealing the head with the sealing liquid twice for 20min each time, oscillating in a shaking table, and then washing, wherein the washing method is the same as the above method, and after washing, pumping the liquid by using a vacuum pump and then washing;
the substitution value calculation is disclosed as: substitution value = OD/(1.65 × m)Resin），mResinIs the mass of the resin.
(4) Addition of the second amino acid, alanine: during synthesis, the process is carried out from the C end to the N end, and the resin is deprotected twice after the first amino acid is added in the step (2);
the deprotection is to add 4 mL of deprotection solution (the volume ratio of piperidine to DMF is 1: 3) into a synthesizer, stir and react for 20min at the temperature of 25-28 ℃, and pump dry by a vacuum pump;
then washing, wherein the washing method and the steps are the same as those in the step (3);
then when the indene detection of the resin is blue, weighing the amounts of alanine, HoBt and DIC of the second amino acid according to the formula 3, wherein the amounts are 217.91mg, 94.591mg and 88.34 mu L respectively, dissolving alanine and HoBt by using 4 mL of DMF, adding the dissolved alanine and HoBt into a synthesizer, directly adding DIC into the synthesizer, and stirring and reacting for 2.5 hours at the temperature of 25-28 ℃; after the reaction is finished, washing the resin according to the method in the step (3), and after the washing is finished, selecting the resin to be colorless through indene detection;
the formula 3 is: the amount of amino acid = the relative molecular mass of the amino acid (proline here) × 2.5 × the mass of the resin (0.3 here) × the substitution value (here, the result of calculation of the substitution value in step (3)).
(5) Addition of subsequent amino acids: the subsequent amino acid adding method is the same as the second amino acid adding process until all the amino acids are added; when the sample is subjected to indene detection, the color development requirement is adjusted, and when the former amino acid is proline, serine and histidine, the color of the sample is reddish brown when the sample is subjected to indene detection.
(6) Cleavage of the polypeptide: cutting polypeptide from resin, firstly carrying out deprotection twice according to the deprotection method in the step (4); washing (same as the step (3)); then cutting is carried out;
the cutting is that a cutting reagent is added into a synthesizer, a stirring column is used for stirring for three hours, then liquid in the synthesizer is pumped into a spherical flask, and DCM is used for washing for 3 times;
the spherical flask is arranged on a rotary evaporator to be evaporated for 1 hour;
adding diethyl ether into the spherical flask, evaporating for 4-6 times, finally adding ethyl acetate, standing on ice for 30min, and obtaining white precipitate as precipitated crude peptide;
centrifuging the ether solution containing crude peptide at 2000rpm for 2min to obtain crude peptide precipitate, and oven drying the crude peptide in oven at 37 deg.C (3 hr for drying);
it is emphasized that the cutting reagent is prepared on site, and the specific composition ratio is as follows: 0.3mL of triple distilled water, 0.3mL of thioanisole, 0.15mL of 1, 2-dithiol, 0.3mL of phenol and 4.95mL of TFA4.
(7) Purification of the crude peptide: RP-HPLC was used to purify the crude peptide by the following purification scheme: acetonitrile, 1% o, TFA =25% -50%; the flow rate is 5min/mL, and the detection wavelength is 228 nm;
the purified refined peptide is the synthetic peptide Z-GP-V2 targeting FAP, and purity detection shows that the purified refined peptide has a purity of more than 95% and can be stored at-20 ℃ for later use.
ESI-MS mass spectrometry was performed on the purified synthetic peptide Z-GP-V2, and the results are shown in FIG. 1. As can be seen from FIG. 1, the molecular weight of the synthetic peptide Z-GP-V2 prepared was as expected.
Example 2
The inventors have conducted specific experimental verification of the anti-tumor angiogenesis effect of the synthetic peptide Z-GP-V2 prepared in example 1, and the related experiments are briefly described as follows.
First, in vitro anti-angiogenesis effect verification
1. Cell scratch test
The effect of synthetic peptide Z-GP-V2 on migration of Human Umbilical Vein Endothelial Cells (HUVECs) was evaluated using a cell scratch assay, the procedures of which are briefly described below.
(1) HUVECs at 1X 105Inoculating the cells with the density of one cell/mL into a 24-well plate, and after the cell plating rate reaches more than 90%, slightly scratching along the mark by using a 200-mu-L gun head;
(2) subsequently, the scratched cells are washed clean by PBS, and Z-GP-V2 is added into each multiple well at different concentrations (100, 25 and 5 mu M) respectively, wherein each concentration is an experimental group, each group comprises 3 multiple wells, and simultaneously, serum-free RPMI 1640 culture medium is used as a negative control group, V2 peptide is used as a positive control group, and Z-GP is used as an irrelevant peptide control group;
the V2 peptide and the Z-GP dipeptide are prepared by referring to the prior art, and are dissolved and diluted to 200 mu M by RPMI 1640 serum-free culture medium before use;
(3) at 37 deg.C, 5% CO2The culture is carried out for 24 hours in a constant temperature incubator, the photographing record is observed under an inverted microscope at the magnification of 40 multiplied times every 12 hours, the scratch healing degree of each time period is calculated according to the following formula, and a histogram is drawn;
calculating the formula: [1- (relative area of 12 hours to 24 hours/relative area of 0 hour) ]. times.100%.
The results of the experiment are shown in FIG. 2. As can be seen from the figure, the synthetic peptide Z-GP-V2 of the invention has the effect of inhibiting the migration of HUVECs, and the healing degree of the wound of the negative control group is 1.3 times that of the test group of 5 mu M Z-GP-V2 at 24 hours, and the result shows that the migration rate of the HUVECs of the test group is slower than that of the negative control group, and the Z-GP-V2 has a good inhibition effect on the migration of the HUVECs. The "wound" healing degree was almost the same for the Z-GP treated group alone as for the negative control group, indicating that Z-GP itself had no significant effect on the migration of HUVECs. As can be seen from the comparison result of the V2 peptide and the synthetic peptide Z-GP-V2 group, the healing degree of the synthetic peptide Z-GP-V2 group is better than that of the V2 peptide group with the prolonging of time, but the synthetic peptide Z-GP-V2 group always has the effect of inhibiting the migration of HUVECs, which indicates that the synthetic peptide Z-GP-V2 has relatively small side effect on the migration of HUVECs, but still maintains the capability of V2 for inhibiting the migration of HUVECs.
2. Cell migration assay
To further verify the effect of Z-GP-V2 in inhibiting HUVECs migration, the inventors performed an experiment (BD Co.) in a Transwell chamber of 8.0 μm, the summary of the experimental procedure is as follows:
(1) HUVECs are subjected to starvation treatment for 12 hours in a serum-free RPMI 1640 culture medium sequentially, are treated for 12 hours in Z-GP-V2 with different drug concentrations (100, 25 and 5 mu M), and are treated for 12 hours by taking a serum-free culture medium as a negative control group, a V2 peptide as a positive control group and a Z-GP unrelated peptide control group, and then cells are collected and resuspended in the serum-free culture medium;
the V2 peptide and the Z-GP dipeptide are prepared by referring to the prior art, and are dissolved and diluted to 200 mu M by RPMI 1640 serum-free culture medium before use;
(2) at 1.5X 105The cells were seeded at a density of 200. mu.L per well in the upper Transwell chamber and 750. mu.L of 10% FBS in RPMI 1640 medium in the lower chamber at 37 ℃ in 5% CO2The culture is carried out for 12 hours in a constant temperature incubator;
(3) after the culture is finished, sucking out the old culture medium, and washing for 2 times by using PBS;
(4) fixing the cells with 3.8% paraformaldehyde for 20 min; after the time, washing with PBS for 2 times;
(5) staining with 0.2% crystal violet for 15 min; after the time, washing with PBS for 5 times;
(6) after cleaning, an appropriate amount of ultrapure water was added to each well, and the mixture was placed under an inverted microscope and observed at a magnification of 40 × to count photographs and draw a histogram.
The results of the experiment are shown in FIG. 3. As can be seen from fig. 3, synthetic peptide Z-GP-V2 had an effect of inhibiting the migration of HUVECs, and the number of migrated cells in the negative control group was 3.4 times that of HUVECs migrated at a concentration of 25 μ M of synthetic peptide Z-GP-V2, with a significant difference (. p. < 0.01), indicating that the migration rate of HUVECs after synthetic peptide Z-GP-V2 treatment was slow, and synthetic peptide Z-GP-V2 had the ability of inhibiting the migration of HUVECs. The number of cell migrations was almost identical for the Z-GP treated group alone compared to the negative control group, again suggesting that Z-GP itself had no significant effect on the migratory capacity of HUVECs. In contrast, the comparison between the V2 peptide group and the synthetic peptide Z-GP-V2 group shows that the synthetic peptide Z-GP-V2 group and the synthetic peptide V2 group have the effect of inhibiting HUVECs from migrating at different concentrations, the number of migrated cells in the two groups is obviously lower than that in the negative control group, and Z-GP-V2 maintains the capacity of V2 in inhibiting endothelial cell migration.
Based on the existing research, the inventor believes that the sequence connected with Z-GP can realize the preliminary positioning function on the basis that Z-GP is used as a substrate of FAP, and the sequence connected with Z-GP can be preliminarily positioned in the tumor stroma because FAP exists in the tumor stroma. In combination with the above in vitro experimental results, it can be seen that the peptide sequence linked to Z-GP (i.e. the synthetic peptide Z-GP-V2 provided by the present invention) still retains better polypeptide activity and still has better anti-tumor effect compared to the original V2 peptide; alternatively, the Z-GP dipeptide substrate itself did not affect the anti-angiogenic effect of the V2 peptide. In view of the lack of targeting property of the initial V2 peptide during the action, the synthetic peptide Z-GP-V2 provided by the invention can better act on a target (vascular endothelial cells), has better improvement effect on better inhibiting the migration of endothelial cells and playing the role of anti-tumor angiogenesis, and simultaneously has better popularization and application significance in view of lower toxic and side effect.
In vivo experimental verification of antitumor activity of synthetic peptide Z-GP-V2
To further examine the antitumor activity of the synthetic peptide Z-GP-V2, further antitumor-related experiments were performed on the synthetic peptide Z-GP-V2 prepared in example 1, as follows.
Experiment of tumor inhibition rate
The experimental procedure was as follows:
(1) the axilla of the right forelimb of each mouse is filled with 1 × 107Tumor cells until the tumor volume of the mouse reaches 50-100 mm3The tumor volumes are randomly grouped into 7 groups, namely a Z-GP-V2 high dose group (0.6 mu M), a Z-GP-V2 low dose group (0.2 mu M), a V2 high dose group (0.6 mu M), a V2 low dose group (0.2 mu M), a Z-GP high dose group (0.6 mu M), a Z-GP low dose group (0.2 mu M) and a physiological saline group (negative control group), wherein each group comprises 5 mice;
when in use, the synthetic peptide Z-GP-V2 (V2 peptide or Z-GP) is directly dissolved in normal saline to prepare corresponding concentration, and is injected according to the injection dosage of 1 mL/Kg;
it should be noted that synthetic peptide Z-GP-V2 (V2 peptide or Z-GP) is dissolved in physiological saline, and needs to be filtered and sterilized, and can be stored at-20 ℃ for standby after being subpackaged after being filtered and sterilized during the period of convenient use;
(2) tail vein injection for 12 days; each group was dosed daily in the morning; mice had free access to food and water during the experiment;
(3) during the experiment, the medicine is taken dailyMeasuring the weight of the mouse, recording and drawing a curve to evaluate the toxic and side effect of the synthetic peptide Z-GP-V2; the long (a) short (b) diameters of the tumors were measured daily at the same time and formulated (calculated as disclosed: V =1/2 × (a × b)2) Calculate tumor volume and plot tumor growth curves; the day after the end of dosing mice were decapitated and tumors were removed and weighed.
The change curve of the mouse body weight is shown in FIG. 4, the change curve of the mouse tumor volume is shown in FIG. 5, and the change curve of the mouse tumor weight is shown in FIG. 6.
As can be seen from FIG. 4, the body weight change of the group to which the synthetic peptide Z-GP-V2 was administered was within the normal range, indicating that the synthetic peptide had no significant toxic side effects. From fig. 5 and fig. 6, it can be seen that the tumor volume and tumor weight of the group administered with Z-GP-V2 are smaller than those of the group with physiological saline as the negative control, and have differences (. p < 0.05), and the high concentration shows better inhibitory effect, and the Z-GP group does not show obvious tumor inhibitory activity, indicating that the synthetic peptide Z-GP-V2 targeting FAP has a certain anti-tumor effect.
Further, in comparison with the synthetic peptide Z-GP-V2 group, although the anti-tumor effect of the Z-GP-V2 treated group was not sufficiently prominent, since the V2 peptide did not target the functional motif of tumor, it was seen from the change in body weight of the mice in fig. 4 that it still had a certain toxic side effect (the increase in body weight of the mice was not significant with the lapse of time), while the Z-GP-V2 experimental group had a certain anti-tumor effect and no significant toxic side effect.
Since the discovery of FAP in 1986, a great deal of research has been carried out on the localization, expression and function of the protein, and preliminary studies suggest that FAP plays an important role in tumor invasion and metastasis, which is associated with 90% of epithelial cell carcinomas, but does not normally express the protein in normal adult tissues, benign and premalignant epithelial lesion tissues, and thus FAP can be a promising target molecule in-vivo immunodiagnosis of tumors.
The dipeptide substrate of FAP is coupled with the targeting V2 peptide with the effect of anti-angiogenesis of Tie2 through an alanine flexible linker, so that the designed synthetic peptide Z-GP-V2 has a double-targeting function, and toxic and side effects caused by non-specificity of the Tie2 selective polypeptide V2 are reduced and reduced by utilizing the high affinity of Z-GP and FAP and the high expression characteristic of FAP at a tumor stroma part, and meanwhile, the anti-tumor angiogenesis effect of the V2 peptide part in the new synthetic peptide is well maintained. Through a series of in vivo and in vitro experiments, the synthetic peptide Z-GP-V2 provided by the invention has no obvious side effect when being applied to anti-angiogenesis tumor formation, and has mature and complete preparation method, thereby having better medical application prospect.
SEQUENCE LISTING
<110> Zhengzhou university
<120> FAP-targeted anti-angiogenic peptide Z-GP-V2 and application thereof
<130>none
<160>1
<170>PatentIn version 3.5
<210>1
<211>11
<212>PRT
<213> synthetic peptide against angiogenesis
<400>1
Gly Pro Ala Ala Asn Leu Leu Met Ala Ala Ser
1 5 10
Claims (3)
1. An anti-angiogenic synthetic peptide Z-GP-V2 targeting FAP, wherein the synthetic peptide has a molecular weight of: 1149.6 Da, which is specifically as follows: Z-Gly-Pro-Ala-Ala-Asn-Leu-Leu-Met-Ala-Ala-Ser; namely Z-GPAANLLMAAS;
the Z-GP is targeting FAP dipeptide substrate N-benzyloxycarbonyl glycine proline.
2. The method for preparing the FAP-targeted anti-angiogenesis synthetic peptide Z-GP-V2 according to claim 1, wherein the peptide is prepared by Fmoc solid-phase peptide synthesis.
3. The use of the FAP-targeting anti-angiogenic peptide Z-GP-V2 in the preparation of an anti-tumor angiogenesis agent according to claim 1, wherein the tumor is a tumor caused by S180 cells.
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