CN116715730A - Self-assembled small molecule peptide and application thereof in anti-angiogenesis - Google Patents

Abstract

The invention discloses a self-assembled small molecule peptide and application thereof in resisting angiogenesis. The amino acid sequence of the self-assembled small molecule peptide is shown as follows: FFKRPPLKRVRMSADAML. Experiments show that the self-assembled small molecular peptide can inhibit vascular endothelial cell proliferation, intestinal vessel formation and cornea angiogenesis, can achieve an anti-tumor effect by inhibiting angiogenesis, has good enzymolysis stability and long half-life period, and can be reserved in a body for a longer time. Therefore, can be used for preparing anti-angiogenesis, anti-tumor or medicaments for treating neovascular eye diseases.

Description

Self-assembled small molecule peptide and application thereof in anti-angiogenesis

Technical Field

The invention relates to the field of biological medicine, in particular to a self-assembled small molecule peptide and application thereof in anti-angiogenesis.

Background

Angiogenesis is the development of new blood vessels from an existing vascular bed and is a complex multi-stage process. Angiogenesis is important for normal physiological processes including, but not limited to, embryo implantation, embryogenesis and development, and wound healing. Angiogenesis is also associated with the progression of non-cancerous diseases, such as neovascular glaucoma, diabetic retinitis, atherosclerosis, obesity, hepatitis, pneumonia, asthma, and tumors.

Although protein drugs such as Ramucirumab, bevacizumab and the like can inhibit receptor signaling by blocking the binding of VEGF and VEGFR2, the protein drugs often generate drug resistance after a period of use, and the protein drugs generally have immunogenicity when injected into human bodies due to larger molecular weight. Polypeptide drugs have been developed to treat a variety of diseases including cancer, immune diseases, metabolic disorders, viral infections, cardiovascular diseases and osteoporosis. In view of its good safety, tolerability and efficacy, it is rapidly becoming a good choice for designing new therapeutic agents, which may also be a major distinguishing factor for polypeptides compared to traditional small molecules. In addition, polypeptides have lower production complexity, and lower production costs, typically approaching that of small molecules, than protein-based biopharmaceuticals. Thus, therapeutic polypeptides are effective tools for the development of anti-angiogenic drugs.

In recent 20 years of clinical research, more than 300 therapeutic peptides for different disease applications are being developed, and more than 80 polypeptide drugs are coming into the market. However, none of these approved polypeptide drugs have substantially the effect of inhibiting angiogenesis. Currently, marine bioactive substances have been attracting attention as effective therapies for treating diseases including cancer and cardiovascular diseases. Some marine-derived bioactive substances have been shown to prevent the formation of new blood vessels, indicating that marine-derived bioactive substances are a rich and promising source for the discovery of new anti-angiogenic drugs.

Disclosure of Invention

The primary purpose of the invention is to overcome the defects and shortcomings of the prior art and provide a self-assembled small molecule peptide.

Another object of the invention is to provide the use of said self-assembled small molecule peptides in the preparation of a medicament for the anti-angiogenic and/or treatment of ocular neovascular diseases.

The invention also aims to provide application of the self-assembled small molecule peptide in preparing antitumor drugs.

The aim of the invention is achieved by the following technical scheme:

a self-assembled small molecule peptide having the amino acid sequence shown below: FFKRPPLKRVRMSADAML.

The self-assembled small molecule peptide can be synthesized by methods conventional in the art, such as Fmoc solid phase synthesis.

The self-assembled small molecule peptide is applied to the preparation of anti-angiogenesis and/or medicaments for treating neovascular eye diseases (ocular neovascular disease, OND).

The anti-angiogenic drugs are drugs for inhibiting proliferation and migration of vascular endothelial cells, inhibiting formation of blood vessels among embryo segments, inhibiting formation of intestinal blood vessels, inhibiting generation of capillary vessels and/or inhibiting generation of branch blood vessels; preferably a drug that inhibits vascular endothelial cell proliferation and/or inhibits intestinal vessel formation.

The blood vessel is a new blood vessel.

The vascular endothelial cells are human umbilical vein endothelial cells.

The embryo is preferably a zebra fish embryo.

The intestinal blood vessel is preferably the intestinal blood vessel of the juvenile zebra fish, and the self-assembled small molecule peptide can inhibit the formation of the intestinal blood vessel of the zebra fish.

The drug for treating the neovascular eye disease is a drug for inhibiting cornea angiogenesis, and the self-assembled small molecule peptide can reduce the number of cornea angiogenesis.

The effective concentration of the self-assembled small molecule peptide is 12.5-250 mu mol/L; preferably 50 to 100. Mu. Mol/L; more preferably 50. Mu. Mol/L.

The application of the self-assembled small molecular peptide in preparing antitumor drugs,

the self-assembled small molecule peptide can inhibit angiogenesis and achieve the anti-tumor effect.

The tumor is tumor cells and/or tumor tissues with highly developed vascular tissues, including liver cancer and the like.

The medicament may contain one or at least two pharmaceutically acceptable carriers.

The carrier is preferably a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant, a lubricant, or the like.

The medicine can be prepared into various dosage forms by adopting a conventional method in the field, including decoction, tablets, pills, capsules, injection (powder injection), granules (medicinal granules), oral liquid and syrup, or tablets, capsules, injection (powder injection), granules and the like prepared by adopting a micro-nano technology.

Compared with the prior art, the invention has the following advantages and effects:

1. the self-assembled small molecule peptide (named as B2F) has the advantages of improved enzymolysis stability and longer half-life, and can be reserved in vivo for a longer time.

2. The B2F peptide has obvious anti-angiogenesis effect on Human Umbilical Vein Endothelial Cells (HUVECs) and zebra fish.

3. The B2F peptide has the anti-angiogenesis effect of inhibiting the migration of Human Umbilical Vein Endothelial Cells (HUVECs) in vitro, and can block the formation of blood vessels under the intestines of zebra fish embryos.

4. The B2F peptide can be chemically synthesized, does not destroy the ecological environment, is easy to realize industrialization, and is a very promising renewable anti-angiogenesis drug resource.

5. The B2F peptide can inhibit cornea angiogenesis, so that the B2F peptide can be used for preparing medicaments for treating neovascular eye diseases.

6. The B2F peptide can inhibit angiogenesis and achieve the anti-tumor effect, so that the B2F peptide can be used for preparing anti-tumor medicines and treating liver cancer.

Drawings

FIG. 1 is a graph showing degradation of B2F peptide of the present invention.

FIG. 2 is a graph showing the inhibition of HUVECs cell growth by the B2F peptide of the present invention in vitro.

FIG. 3 is a graph showing the inhibition of HUVECs cell migration by the B2F peptide of the present invention in vitro.

FIG. 4 is a graph showing the inhibition of angiogenesis of zebra fish embryos by the B2F peptide of the present invention in vivo.

FIG. 5 is a graph showing the effect of B2F peptide on corneal angiogenesis in the present invention; wherein A is a corneal vessel treated by the B2F peptide; b is the inverse of A.

FIG. 6 is a TEM result diagram of B2F peptide and B2 peptide in the present invention; wherein A is a TEM image of the B2F peptide; b is a TEM image of the B2 peptide.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.

EXAMPLE 1 Synthesis of self-assembled B2F peptides

The amino acid sequence of the B2F peptide in the present invention is as follows: FFKRPPLKRVRMSADAML the Fmoc solid phase synthesis method was adopted by Shanghai Yao Biotechnology Co.Ltd. The main involved synthetic raw materials, related reagents, instruments and equipment and the synthetic process are briefly described as follows:

1.1 raw materials for synthesis and related reagents: (1) protecting amino acid raw materials: fmoc protected amino acids; (2) condensing reagent: o-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), N, N-Diisopropylethylamine (DIEA); (3) solvent: n, N-Dimethylformamide (DMF), dichloromethane (DCM), methanol, acetonitrile; (4) resin: 2-Chlorotrityl Chloride resin having a substitution degree of 1.1 mmol/g; (5) deprotection reagent: piperidine; (6) detection reagent: phenol reagent, pyridine reagent, ninhydrin reagent; (7) cutting reagent: trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1, 2-Ethanedithiol (EDT), anhydrous diethyl ether; (8) nitrogen gas; (9) a precision electronic balance.

1.2 instrumentation: (1) a twelve-channel semiautomatic polypeptide synthesizer; (2) a high performance liquid chromatograph; (3) a freeze dryer; (4) and (5) a centrifugal machine.

1.3 synthetic procedure:

1. swelling of the resin: resin 2-Chlorotrityl Chloride Resin was placed in a reaction tube and DMF (15 ml/g, i.e., 15ml solvent per gram of resin, the same applies below) was added and shaken for 60min.

2. The first amino acid: the solvent was filtered off with suction through a sand core, 3 times the molar amount of Fmoc-Cys (Trt) -OH amino acid (first amino acid at C-terminus) was added, 10 times the molar amount of DIEA was added, and finally DMF was added for dissolution and shaking for 30min. And (5) sealing with methanol, and standing for 30min.

3. Deprotection: DMF was removed, a 20% (v/v) piperidine-containing DMF solution (15 ml/g) was added, allowed to stand for 5min, and a 20% (v/v) piperidine-containing DMF solution (15 ml/g) was removed and allowed to stand for 15min.

4. And (3) detection: pumping off piperidine solution, taking more than ten deprotected resin, washing with ethanol for three times, adding ninhydrin reagent, KCN solution and phenol solution, heating at 105-110 ℃ for 5min, and turning into blue positive reaction.

5. Washing: the mixture was washed twice with DMF (10 ml/g), twice with methanol (10 ml/g) and twice with DMF (10 ml/g).

6. Condensation: 3 times molar amount of Fmoc protected amino acid, 3 times molar amount of HBTU, 10 times molar amount of DIEA and finally DMF were added for dissolution and shaking for 45min.

7. And (3) detection: taking more than ten grains of the condensed resin, washing the resin with ethanol for three times, adding one drop of ninhydrin reagent, pyridine and phenol solution, heating the mixture for 5 minutes at the temperature of 105-110 ℃, and taking colorless negative reaction.

8. Washing: washing with DMF (10 ml/g) once, methanol (10 ml/g) twice, and DMF (10 ml/g) twice.

9. Repeating the third to eighth steps, and linking the amino acids in the sequence from right to left until the last amino acid.

10. The resin was washed with DMF (10 ml/g) twice, DCM (10 ml/g) three times, methanol (10 ml/g) four times and dried for 10min as follows.

11. Cutting: preparing a cutting fluid: 94.5% TFA, 2.5% water, 2.5% EDT, 1% TIS (mass fraction); then, a cutting liquid (10 ml/g) was added to cut the resin for 180 minutes.

12. Drying and washing: drying the cut lysate with nitrogen as much as possible, precipitating diethyl ether, centrifuging to remove supernatant, washing precipitate with diethyl ether for six times, and volatilizing at normal temperature to obtain intermediate.

13. Oxidizing: after the dried intermediate is completely dissolved by using water/acetonitrile, 5% (v/v) dimethyl sulfoxide (DMSO) is added, pH=8-9 is added, and the mixture is magnetically stirred at room temperature for 8 hours, monitoring is carried out by using an Ellman reagent until the oxidation is complete, and freeze-drying is carried out to obtain an oxidized crude product.

14. Purifying and preparing: (1) taking a little crude product, H ₂ O/acetonitrile is dissolved; (2) taking a small amount of samples, and analyzing and judging the peak time corresponding to the target peak on an HPLC analysis instrument; (3) preparation system using C18 reverse phase chromatography: wavelength: 220nm; flow rate: 15ml/min; volume (inj.vol): 20mL; column temperature: 25 ℃; eluting: buffer A, 0.1% (v/v) TFA in water; buffer B: acetonitrile solution containing 0.1% (v/v) TFA; collecting a target peak solution; (4) a few target peak solutions were taken with a 1.5ml centrifuge tube for mass spectrometry confirmation and purity detection.

15. And freeze-drying the qualified target peak solution to obtain a finished product.

16. And (3) identification: taking a small amount of the finished polypeptide, and performing molecular weight identification of MS and purity identification of HPLC analysis.

17. The B2F peptide was packed in a white powder form, and stored at-20 ℃.

Example 2 comparison of stability of self-assembled B2F peptides and B2 peptides

2.1 Experimental materials

Reagent: the self-assembled B2F peptide and B2 peptide (amino acid sequence: KGKFKRPPLKRVRMSADAML) prepared in example 1 were synthesized by Shanghai Yao Biotechnology Co., ltd.) in the same manner as in example 1.

2.2 Experimental methods

Uniformly mixing serum obtained after low-speed centrifugation of blood of healthy volunteers with the B2F peptide prepared in example 1 in a ratio of 9:1 (mL: mg) to make the concentration of polypeptide in the polypeptide-serum mixture be 1mg/mL; then placing the polypeptide-serum mixture in a 37 ℃ water bath for incubation for different times, wherein details are shown in table 1, and after a preset time is reached, adding pure trifluoroacetic acid into each sample respectively to ensure that the final concentration of the trifluoroacetic acid in the mixture is 10% (v/v); centrifuging at 13000rpm for 10min after shaking, and collecting 10 μl of supernatant in HPLC sample loading bottle; HPLC-UV analysis (apparatus model Waters ACQUITY Arc Bio) was performed using a C18 analytical column (Thermo Acclaim 300C 18) with an ultraviolet wavelength of 220nm, a loading of 20. Mu.L, a flow rate of 0.5mL/min, and a column temperature of 25℃was set as described in Table 1 below. The target peak was determined from the retention time, its peak area was recorded, and a degradation curve was made. The experiment was set up in triplicate with B2 peptide as control.

TABLE 1

Time (min)	Buffer A (0.1% (v/v) TFA aqueous solution)	Buffer B (acetonitrile solution containing 0.1% (v/v) TFA)
			0min	95％	5％
27min	75％	25％
			28min	5％	95％
33min	5％	95％
			40min	95％	5％
45min	95％	5％

2.3 experimental results

The results are shown in fig. 1, the in vitro degradation curve of self-assembled B2F is more gentle than the degradation of B2, demonstrating that self-assembled B2F has higher stability than B2 and longer half-life.

Example 3 Effect of self-assembled B2F peptides on HUVECs cell proliferation in vitro

3.1 Experimental materials

And (3) cells: since the object of the present invention is to inhibit angiogenesis, it is first of all in HUVEC cell lines (purchased from Shanghai cell Bank).

Reagent: as in example 2.

3.2 Experimental methods

CCK8 proliferation assay: HUVEC cells were cultured to log phase in DMEM medium supplemented with 10% (v/v) fetal bovine serum, cells were collected by digestion, plated in 96-well plates uniformly at a cell density of 3000/well, starved with serum-free DMEM medium for 12 hours after 24 hours of adherence, and then replaced with DMEM medium supplemented with 0.5% (v/v) fetal bovine serum and treated with B2F peptide for 48 hours with 6 duplicate wells per concentration gradient; wherein, the final concentration of the B2F peptide is 12.5, 25 and 50 mu M respectively, the blank control is not added with the B2F peptide, and the control is added with the B2 peptide. After 48 hours, the culture medium was discarded, the DMEM medium containing 10% (v/v) CCK8 solution was changed, incubated in a 37℃incubator for 60 minutes, and absorbance at 450nM wavelength was measured under an microplate reader.

3.3 experimental results

The results are shown in fig. 2, and the results of the cell proliferation experiments show that the B2F peptide has a remarkable inhibition effect on the proliferation of HUVEC cells and has concentration dependence.

Example 4 Effect of self-assembled B2F peptides on HUVECs cell migration in vitro

4.1 Experimental materials

And (3) cells: since the object of the present invention is to inhibit angiogenesis, it is first of all in HUVEC cell lines (purchased from Shanghai cell Bank).

Reagent: 25. Mu.M, 50. Mu.M B2F peptide, 4% paraformaldehyde and 0.1% crystal violet.

4.2 Experimental methods

Cell migration experimental method: cell migration was tested in a transwell chamber with a pore size of 8 μm. HUVECs cells were resuspended in serum-free DMEM medium and transferred to the upper chamber of a transwell 24-well plate. DMEM cell culture medium containing 10% (v/v) Fetal Bovine Serum (FBS) and B2F peptide (final concentration 25 μm, 50 μm) was then added to the upper chamber of the transwell chamber, and 3 duplicate wells were set per concentration gradient to blank without B2F peptide. After 24 hours of treatment, the cells were fixed with a 4% paraformaldehyde fixative, stained with 0.1% crystal violet, gently rubbed with a cotton swab against the upper surface of the membrane within the transwell cells, and observed and imaged using an inverted microscope.

4.3 experimental results

The results of 3 duplicate wells are shown in figure 3, where B2F peptide significantly reduced the number of HUVECs cells migrating through the membrane in a Transwell cell migration experiment, indicating that B2F peptide can inhibit HUVECs migration.

Example 5 Effect of self-assembled B2F peptides on intestinal angiogenesis in zebra fish embryos

5.1 Experimental materials

Animals: transgenic zebra fish (flk 1: EGFP), purchased from the national zebra fish resource center (s 843Tg/+, ID: ZDB-TGCONSTRCT-070117-47).

Reagent: B2F peptide.

5.2 Experimental methods

Transgenic zebra fish with blood vessels marked by green fluorescence are spawned one night in advance, and fish eggs are collected the next day. The vitelline membrane was broken at 24 hours using a membrane breaker pronase (2 mg/mL) (available from Shanghai Milin Biochemical technologies Co., ltd., cat. No. P916050-100 g). B2F peptide was diluted to different concentrations (0, 10, 50, 250 μΜ) with culture water and microinjected under a dissecting microscope, 5nL of different concentrations of B2F peptide solution was microinjected into the abdominal cavity of zebrafish embryos, and the experiment was set up in triplicate with no B2F peptide added as a blank. At 72hpf (i.e., 72 hours after fertilization), images of zebra fish angiogenesis were taken using a split fluorescence microscope.

5.3 experimental results

As a result, as shown in fig. 4, a significant decrease in the number of capillaries and branched vessels was observed after treatment with B2F peptide (50 μm, 250 μm) compared to the blank group. Demonstrating that B2F peptide was significantly inhibited in intestinal vessel (SIV) formation in young zebra fish by microinjection into young zebra fish (24 hpf) and was concentration dependent after culturing to 72 hpf.

Example 6 Effect of self-assembled B2F and B2 peptides on corneal angiogenesis

6.1 Experimental materials

Animals: BALB/cJGpt mice (N000020), 6 week old, male, purchased from Guangdong Kangdong biotechnology.

Reagent: B2F peptide, B2 peptide, chloral hydrate (CE 202-100g, purchased from Shanghai Jiding Biotechnology Co., ltd.), tetracaine hydrochloride (IT 3760, beijing Soy Bao technology Co., ltd.), naOH (I821551-50 ml, purchased from Guangzhou Jixian technology Co., ltd.), physiological saline (MA 0083, meiluno/Mei Lun organism).

6.2 Experimental methods

BALB/cJGpt mice at 6 weeks of age were given intraperitoneal injection of 4% chloral hydrate (0.2 mL/20 g) to induce general anesthesia. After fixation in the supine position, the beard and eyelash were cut off, and 1% (w/v) oxybutynin hydrochloride solution was added dropwise to the eyes to anesthetize the eyes of the mice. A single-layer round filter paper with the diameter of 2mm is immersed in a 1mol/L NaOH solution for 30s to reach a saturated state, and the water-absorbing paper is applied to the center of the right eye cornea of a mouse for 30s after absorbing excessive liquid. The filter paper sheet was removed, and the conjunctival sac was immediately rinsed with physiological saline for 1min. 2. Mu.L of physiological saline was dropped on a filter paper sheet by a pipette and placed on the left eye of a mouse, and the rest steps were the same as before, as an alkali burn control.

After the model is built, subconjunctival injection administration can be carried out, and the day of model building is the 0 th day of administration. The 12 mice were randomly divided into four groups: blank (no treatment, n=3), model (NaOH burning ocular surface, n=3); b2 dosing group (physiological saline dissolution, concentration 50 μm, n=3); B2F-administered group (physiological saline solution, concentration 50 μm, n=3). The injection volumes were 25. Mu.L each.

6.3 experimental results

As a result, as shown in fig. 5, the corneal blood vessels of the model group were significantly increased compared to the blank group, and a significant decrease in the number of corneal angiogenesis was observed after treatment with B2 peptide and B2F peptide (50 μm) (fig. 5A), indicating that the treatment of corneal blood vessel neogenesis disease was enabled by the B2 peptide and B2F peptide. Fig. 5B is an inverse view of fig. 5A to better observe the growth of blood vessels.

EXAMPLE 7 Transmission Electron microscopy of self-assembled B2F and B2 peptides

7.1 Experimental materials

Reagent: B2F peptide, B2 peptide.

7.2 Experimental methods

Respectively diluting the two polypeptides to 0.1mg/ml, dripping the two polypeptides into a copper mesh matched with an instrument, and air-drying at room temperature; the polypeptide-loaded copper mesh was placed in a TEM instrument (instrument model TECNAL G2 Spirit TWIN) and observed.

7.3 experimental results

As a result, as shown in fig. 6, the B2F peptide formed a regular spherical structure (fig. 6A), while the electron microscope image of the B2 peptide was less regular (fig. 6B), indicating that the B2F peptide formed a stable nanosphere-shaped self-assembled structure.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A self-assembled small molecule peptide, characterized by the amino acid sequence as follows: FFKRPPLKRVRMSADAML.

2. Use of the self-assembled small molecule peptide of claim 1 for the manufacture of a medicament for anti-angiogenic and/or treating ocular neovascular disorders.

3. The use according to claim 2, characterized in that:

the anti-angiogenic drugs are drugs for inhibiting proliferation and migration of vascular endothelial cells, inhibiting formation of blood vessels among embryo segments, inhibiting formation of intestinal blood vessels, inhibiting generation of capillary vessels and/or inhibiting generation of branch blood vessels;

the medicine for treating the neovascular eye disease is a medicine for inhibiting cornea angiogenesis.

4. A use according to claim3, characterized in that:

the vascular endothelial cells are human umbilical vein endothelial cells;

the embryo is a zebra fish embryo;

the intestinal blood vessel is of juvenile zebra fish.

5. The use according to claim 2, characterized in that: the effective concentration of the self-assembled small molecule peptide is 12.5-250 mu mol/L.

6. The use according to claim 5, characterized in that: the effective concentration of the self-assembled small molecule peptide is 50-100 mu mol/L.

7. The use according to claim 6, characterized in that: the effective concentration of the self-assembled small molecule peptide is 50 mu mol/L.

8. The use of the self-assembled small molecule peptide of claim 1 in the preparation of an antitumor drug.

9. The use according to claim 8, characterized in that: the tumor is tumor cells and/or tumor tissues with highly developed vascular tissues.

10. The use according to claim 9, characterized in that: the tumor is liver cancer.