CN117903281A - Polypeptide for inhibiting tumor metastasis and design and preparation method thereof - Google Patents
Polypeptide for inhibiting tumor metastasis and design and preparation method thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a polypeptide for inhibiting tumor metastasis and a design preparation method thereof. In the present invention, the polypeptide comprises the R328-E342 and/or K350-Q361 sequences of the amyloid precursor protein APP; the polypeptide takes R328-E342 at the rear section of H1 spiral and K350-Q361 at the front end of H2 spiral as female parent; the polypeptide adopts disulfide bonds to carry out chemical binding for deconstruction stabilization. The polypeptide prepared by binding the polypeptide through chemical bonds to increase the stability of the polypeptide can obviously inhibit the necrotic apoptosis of endothelial cells induced by tumors and reduce the migration of tumor cells across endothelial cell layers. The polypeptide prepared by the invention can obviously inhibit the formation of lung metastasis of a model mouse under the treatment dosage of 10mg/kg, improve the survival rate of the mouse, and can be used as a candidate drug for inhibiting tumor metastasis.
Description
Technical Field
The invention belongs to the technical field of tumor metastasis inhibition, and particularly relates to a polypeptide for inhibiting tumor metastasis and a design preparation method thereof.
Background
Tumor metastasis is a continuous, multi-step, active process in which tumor cells actively enter the circulatory system and escape through the vascular endothelial barrier to eventually colonize to form a secondary tumor. The escape of circulating tumor cells from the endothelial barrier is one of the important links in tumor metastasis. Individual tumor cells are relatively fragile in the circulatory system, and their malignancy is largely dependent on their efficiency of escape through the endothelial barrier.
Amyloid precursor protein (Amyloid precursor protein, APP) on the surface of tumor cells has been found to be capable of promoting the escape of endothelial barrier by stimulating receptor interacting protein 1 (receptor-INTERACTING PROTEIN, ripk1) by binding to vascular endothelial cell surface receptor DR6 (DR 6), followed by formation of necrotic corpuscles (complex IIb) composed of RIPK1, FADD, casp8, RIPK3 and mixed lineage kinase domain-like pseudokinase (MLKL), thereby inducing necrotic apoptosis of vascular endothelial cells, achieving perforation of vascular wall, and promoting the escape of endothelial barrier by tumor cells (see non-patent document 1).
The DR6 and APP interaction areas belong to extracellular targets, the inhibitor can play a role without entering cytoplasm, and the main molecule interaction area is relatively large, so that the inhibitor is very suitable for the polypeptide medicine to play a role. Although DR6 antibody treatment can fully inhibit the interaction between DR6 and APP, the DR6 antibody has the defects of large molecular weight, short biological half-life, certain hepatotoxicity and the like, and influences the normal physiological functions of vascular endothelial cells after the DR6 is completely blocked, so that the DR6 antibody is difficult to develop clinical application. At present, no drug is marketed for inhibitors of DR6/APP interaction targets.
Disclosure of Invention
The invention aims at: in order to solve the above-mentioned problems, a polypeptide for inhibiting tumor metastasis and its preparation method are provided.
The technical scheme adopted by the invention is as follows: a polypeptide that inhibits tumor metastasis, the polypeptide comprising the R328-E342 and/or K350-Q361 sequence of amyloid precursor protein APP;
the polypeptide takes R328-E342 at the rear section of H1 spiral and K350-Q361 at the front end of H2 spiral as female parent;
The polypeptide adopts disulfide bonds to carry out chemical binding for deconstruction stabilization.
In a preferred embodiment, the polypeptide is chemically bound to increase its stability.
In a preferred embodiment, the chemical binding means includes, but is not limited to, disulfide bonds, amide bonds, thiol-ene reactions, huisgen cyclization, halogen aromatic substitution, and the like.
In a preferred embodiment, the polypeptide is for use as a medicament for inhibiting tumor metastasis.
In a preferred embodiment, the polypeptide reduces metastasis of tumor cells by inhibiting tumor cell-induced endothelial cell necrosis.
In a preferred embodiment, the tumor includes, but is not limited to: melanoma, breast cancer, lung cancer or other primary tumors.
In a preferred embodiment, a pharmaceutical combination of polypeptides for inhibiting tumor metastasis, comprising the polypeptides and a pharmaceutically acceptable carrier or excipient.
In a preferred embodiment, the pharmaceutical composition is any one of an external preparation, an oral preparation or an injection preparation.
In a preferred embodiment, the preparation method comprises the steps of: the method is synthesized by adopting a solid-phase chemical synthesis process of an FMOC method, and oxidation reaction is carried out on N-terminal cysteine and C-terminal cysteine to complete disulfide bond cyclization, and the purity is more than 95% after purification by preparative high performance liquid chromatography.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
In the invention, the polypeptide prepared by the invention can obviously inhibit the necrotic apoptosis of endothelial cells induced by tumors and reduce the migration of tumor cells across endothelial cell layers. The polypeptide prepared by the invention can obviously inhibit the formation of lung metastasis of a model mouse at a therapeutic dose of 10mg/kg, improves the survival rate of the mouse, has a certain clinical transformation potential, so that the polypeptide prepared by the invention plays a more efficient and stable role in the treatment and inhibition processes of cancers, and improves the treatment and alleviation effects of the cancers.
Drawings
FIG. 1 is an analytical High Performance Liquid Chromatography (HPLC) of the polypeptide DAI-01 of the present invention, wherein A is a linear sequence synthesized directly by FMOC method, and B is a cyclic peptide DAI-01 generated after disulfide bond stabilization.
FIG. 2 shows the effect of the polypeptide DAI-O1 of the present invention on expression of endothelial cell necrosis-related protein induced by tumor cells.
FIG. 3 is a fluorescent image of necrotic apoptosis of endothelial cells induced by the polypeptide DAI-01 of the present invention.
FIG. 4 is a fluorescent image of the polypeptide DAI-01 of the present invention inhibiting the level of tumor cell migration across the endothelial cell layer.
FIG. 5 is a general diagram of the inhibition of lung metastasis formation in a model animal by the polypeptide DAI-01 of the invention.
FIG. 6 is a fluorescence image of a model animal in which the polypeptide DAI-01 of the present invention inhibits the formation of lung metastases.
FIG. 7 shows survival curves of the subject polypeptide DAI-01 treated and control model animals.
FIG. 8 shows the results of inhibition of lung metastasis of breast cancer in an animal model by treatment with the polypeptide DAI-01 of the present invention.
FIG. 9 shows the results of an antitrypsin hydrolysis test of the polypeptide DAI-01 of the present invention and its pre-cyclization sequence.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
With reference to figures 1-5 of the drawings,
Example one, synthesis and preparation of DAI-01
The invention analyzes the side chain interaction of DR6/APP in a eutectic structure (PDBID=4YN0), and discovers that the interaction of DR6 part is mainly provided by a first cysteine-rich motif, and the motif has a complex tertiary structure; whereas the APP part interaction is concentrated in two sections of antiparallel spiral structures, namely the rear section R328-E342 of the H1 spiral and the front end K350-Q361 of the H2 spiral, the three-level structure of the part is clear. The polypeptide drugs have high selectivity and high affinity similar to antibodies, and the smaller molecular volume of the polypeptide drugs has lower influence on other functions of the non-binding domain of the target molecules. The invention designs a competitive inhibition polypeptide drug with APP/DR6 by taking the two antiparallel spiral structures as female parents, and adopts disulfide bonds for chemical binding for deconstruction stabilization in order to further stabilize a polypeptide secondary structure. For convenience of description, the cyclic peptide was designated DAI-01. The specific sequence is as follows:
The polypeptide DAI-01 is synthesized by adopting an FMOC method solid-phase chemical synthesis process, disulfide bond cyclization is completed by carrying out oxidation reaction on N-terminal cysteine and C-terminal cysteine, the purity is more than 95% after purification by preparative high performance liquid chromatography, the analytical high performance liquid chromatography is shown in the attached figure 1, and the synthesis yield is about 100mg.
Example II, DAI-01 inhibits tumor cell-induced necrotic apoptosis of endothelial cells (Western Blotting)
A polypeptide that inhibits tumor metastasis, the polypeptide comprising the R328-E342 and/or K350-Q361 sequence of amyloid precursor protein APP;
the polypeptide takes R328-E342 at the rear section of H1 spiral and K350-Q361 at the front end of H2 spiral as female parent;
The polypeptide adopts disulfide bonds to carry out chemical binding for deconstruction stabilization.
Endothelial cells HUVEC in logarithmic growth phase were collected by digestion, 1X 10≡5 cells were counted, resuspended in 1mL of DMEM medium containing 10% FBS, inoculated into six-well plates, and cultured in a carbon dioxide incubator at 37℃for 24 hours.
Tumor cells in the logarithmic growth phase are collected by digestion, 5X 10-4 tumor cells are counted, a control group is prepared into suspension by using 1mL of serum-free DMEM medium and added into endothelial cells of the removed medium, and an experimental group is prepared into suspension by using serum-free DMEM medium containing polypeptides with different concentrations and then added into the endothelial cells of the removed medium. After further incubation for 24 hours, the cell samples were lysed using RIPA by washing with PBS twice, and the expression levels of the necroptosis-related proteins were detected by Western Blotting after preparation.
Western Blotting results showed (FIG. 2) that DAI-01 inhibited the expression of the apoptosis-related protein RIP3 in a dose-dependent manner, but was able to inhibit its phosphorylation without affecting MKLK expression, which was directly related to necrotic apoptosis. The final conclusion was that DAI-01 inhibited the expression of endothelial cell necrotic apoptosis-related proteins induced by tumor cells under co-culture conditions in a dose-dependent manner.
Example III DAI-01 inhibits tumor cell-induced necrotic apoptosis of endothelial cells (fluorescence imaging)
Endothelial cells HUVEC in logarithmic growth phase were collected by digestion, 1X 10≡5 cells were counted, resuspended in 1mL of DMEM medium containing 10% FBS, inoculated into six-well plates, and cultured in a carbon dioxide incubator at 37℃for 24 hours.
The culture medium is prepared into suspension and added into endothelial cells removed from the culture medium, and the experimental group adopts serum-free DMEM culture medium containing 10 mu M polypeptide to prepare suspension and then adds into endothelial cells removed from the culture medium. After further incubation for 24 hours, the cells were stained with PI by washing twice with PBS and necrotic cells stained with red fluorescence.
The result of cell fluorescence imaging shows (figure 3), 10 mu M DAI-01 can obviously reduce the cell ratio containing red fluorescence, which suggests that DAI-01 can obviously inhibit the necrotic apoptosis of endothelial cells induced by tumor cells.
Example four, tumor cell migration experiments across endothelial cell layers
Endothelial cells HUVEC in logarithmic growth phase were inoculated in a TRANS WELL upper chamber at 1X 10A 5 and cultured in a 37℃carbon dioxide incubator to a cell density of about 90%. Then 2X 10-5 tumor cells were added to the upper chamber and 1ml of DMEM medium containing 10% FBS was added to the lower chamber, the polypeptide was then added to the upper chamber, and the blank group was added with equal volume of PBS as a control. After 48 hours of incubation, the lower chamber was photographed with a fluorescence microscope.
The migration experiment result shows (figure 4), DAI-01 treatment can obviously reduce the entry of tumor cells with green fluorescent markers into a lower chamber and inhibit the migration capacity of the tumor cells across endothelial cell layers.
Fifth example, anti-tumor metastasis animal experiment (lung general illumination)
Melanoma cells are prepared into single cell suspensions, and C57 mice of 10 weeks of age are injected through tail veins according to the number of 1X 10 to the power of 6 cells to construct a metastasis model of the melanoma.
Model mice were randomized and were subjected to DAI-01 intraperitoneal injection (10 mg/kg/day, saline-injected group as untreated control). After culturing for three weeks, mice were sacrificed, organs were dissected and isolated, and lung tissue was photographed in general, and the results are shown in fig. 5. The results showed that the lungs of the control group of mice injected with physiological saline formed numerous macroscopic melanoma-like metastases, while the lung surfaces of the DAI-01 treated group of mice were barely visible with visible metastases.
Example six anti-tumor metastasis animal experiment (Living imaging)
The blank plasmid containing Luciferase is stably transferred into a melanoma cell line B16 through slow virus, puromycin is screened for 4 generations, and then is digested and harvested to prepare single cell suspension, and C57 mice with the age of 10 weeks are injected through tail vein according to the number of 1X 10-6 to construct a transfer model of melanoma.
After the model mice were randomly grouped, DAI-01 intraperitoneal injection treatment (10 mg/kg/day, once a day) was performed, and a physiological saline injection group was used as an untreated control. Culturing for 3 weeks, anesthetizing the animals, injecting D-luciferin into tail vein, performing small animal imaging analysis after 10 minutes, and judging the tumor cell distribution condition through fluorescence intensity.
As can be seen from the results of in vivo imaging of animals (FIG. 6), the control group injected with physiological saline showed significant lung development at three weeks, whereas the DAI-01 treated mice did not see significant metastases in the lung and other organs except for subcutaneous in situ tumor formation in situ.
Seventh embodiment, anti-tumor metastasis animal experiment (survival rate of mice)
Melanoma cells are prepared into single cell suspensions, and C57 mice of 10 weeks of age are injected through tail veins according to the number of 1X 10 to the power of 6 cells to construct a metastasis model of the melanoma.
Model mice were randomized and were subjected to DAI-01 intraperitoneal injection (10 mg/kg/day, saline-injected group as untreated control). Continuous culture, observation and record of death time of mice and drawing of survival curve.
As can be seen from the survival curve results, the untreated control mice began to die from individuals 27 days after molding, and all individuals died at 33 days; DAI-01 treatment group, no individual deaths occurred until the observation time (60 days after molding).
Example eight anti-tumor metastasis animal experiment (breast cancer lung metastasis model tissue fluorescence imaging)
The blank plasmid containing RFP (red fluorescent protein) is stably transferred into a murine breast cancer cell line 4T1 through slow virus, puromycin is screened for 4 generations, and then is digested and harvested to prepare single cell suspension, and C57 mice with the age of 10 weeks are injected through tail vein according to the number of 1X 10-6 to construct a lung metastasis model of breast cancer.
After the model mice were randomly grouped, DAI-01 intraperitoneal injection treatment (10 mg/kg/day, once a day) was performed, and a physiological saline injection group was used as an untreated control. Culturing for 2 weeks, killing animals, dissecting and stripping the lung, performing fluorescence imaging analysis, and judging the distribution of tumor cells through fluorescence intensity.
As can be seen from the results of fluorescence imaging (FIG. 8), the control group injected with physiological saline had multiple metastases carrying high-intensity red fluorescence in the lungs at two weeks, whereas DAI-01 treated mice had few metastases with lower red fluorescence in the lungs.
Example nine proteolytic stability of polypeptide
PBS (ph=7.4) solubilized polypeptide to a final concentration of 1mM. Trypsin was dissolved in PBS (2 mM CaCl2, ph=7.4) to a final concentration of 5 μg/ml. The peptide solution (1 ml) was incubated with trypsin solution (10 μl) at 25deg.C. Samples were taken at various time points and the reaction was quenched by the addition of 20. Mu.L of hydrochloric acid (1M). Solutions of tryptic peptide fragments were monitored at various time points using HPLC to determine the proportion of protease degradation.
As can be seen from the results of the examples, the linear polypeptides have poor enzymolysis resistance, and about 21% of the remaining polypeptides have half lives of less than ten minutes after trypsin treatment for 10 minutes. The enzymolysis resistance of the cyclized DAI-01 is greatly improved, and the pancreas protein treatment half-life period is about 120 minutes
From the above test data, it can be seen that: in the invention, the polypeptide prepared by the invention can obviously inhibit the necrotic apoptosis of endothelial cells induced by tumors and reduce the migration of tumor cells across endothelial cell layers. The polypeptide prepared by the invention can obviously inhibit the formation of lung metastasis of a model mouse at a therapeutic dose of 10mg/kg, improves the survival rate of the mouse, has a certain clinical transformation potential, so that the polypeptide prepared by the invention plays a more efficient and stable role in the treatment and inhibition processes of cancers, and improves the treatment and alleviation effects of the cancers.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A polypeptide that inhibits tumor metastasis, characterized in that: the polypeptide comprises R328-E342 and/or K350-Q361 sequences of amyloid precursor protein APP;
the polypeptide takes R328-E342 at the rear section of H1 spiral and K350-Q361 at the front end of H2 spiral as female parent;
The polypeptide adopts disulfide bonds to carry out chemical binding for deconstruction stabilization.
2. The polypeptide for inhibiting tumor metastasis and its design and preparation process as claimed in claim 1, wherein: the polypeptide is bound by chemical bonds to increase the stability of the polypeptide.
3. The polypeptide for inhibiting tumor metastasis and its design and preparation process as claimed in claim 2, wherein: the chemical binding mode comprises the following steps: disulfide bonds, amide bonds, thiol-ene reactions, huisgen cyclizations, and halogen aromatic substitutions.
4. Use of a polypeptide for inhibiting tumor metastasis according to claim 1, wherein: the application of the polypeptide as a medicament for inhibiting tumor metastasis.
5. Use of a polypeptide for inhibiting tumor metastasis according to claim 1, wherein: the polypeptide reduces metastasis of tumor cells by inhibiting tumor cell-induced endothelial cell necrosis.
6. The polypeptide for inhibiting tumor metastasis and its design and preparation method according to claim 4, wherein: the tumor comprises: melanoma, breast cancer, lung cancer or other primary tumors.
7. A polypeptide pharmaceutical composition for inhibiting tumor metastasis, characterized in that: the pharmaceutical composition comprises the polypeptide of any one of claims 1 and a pharmaceutically acceptable carrier or excipient.
8. The polypeptide for inhibiting tumor metastasis and its design and preparation method according to claim 7, wherein: the medicine composition is any one of an external preparation, an oral preparation or an injection preparation.
9. A method for producing a polypeptide for inhibiting tumor metastasis according to claim 1, wherein: the preparation method comprises the following steps: the method is synthesized by adopting a solid-phase chemical synthesis process of an FMOC method, and oxidation reaction is carried out on N-terminal cysteine and C-terminal cysteine to complete disulfide bond cyclization, and the purity is more than 95% after purification by preparative high performance liquid chromatography.
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