Application of mimic polypeptide containing fibrinogen RGD module in preparation of medicine for preventing and / or treating Parkinson's disease

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of simulated polypeptide containing a fibrinogen RGD module in preparation of a medicine for preventing and / or treating Parkinson's disease. The sequence of the mimic polypeptide containing the fibrinogen RGD module disclosed by the invention is as follows: Gly-Arg-Gly-Asp-Ser-Pro-Lys-Asn-Pro-Ser-Cys. The invention further discloses a preparation method of the mimic polypeptide containing the fibrinogen RGD module. The applicant of the invention finds that FG has abnormal accumulation in a nigra compact part of a PD mouse, and the FG excessively aggregated in the brain can promote abnormal aggregation of alpha-syn of dopaminergic neurons through mediation of an alpha v beta 3 integrin receptor. The mimic polypeptide provided by the invention can effectively inhibit alpha-syn abnormal aggregation and dopaminergic neuron death caused by abnormally accumulated FG in the brain to dopaminergic neuron, improve the athletic ability of MPTP modeled PD mice, and provide a new target and idea for PD treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a mimetic polypeptide containing a fibrinogen RGD module in the preparation of drugs for preventing and / or treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD), as the second most common neurodegenerative disease, is mainly pathologically characterized by the destruction of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies containing α-synuclein (α-syn). At present, there is no effective radical cure or therapeutic drug for PD. Therefore, it has great clinical significance to provide more potential therapeutic targets and treatment strategies for PD through the study of the PD pathological mechanism.

[0003] In PD, the pathological mechanism of abnormal aggregation of α-syn is very complex. Some studies have shown that the disruption of the blood-brain barrier in PD is also closely related to the abnormal aggregation of α-syn. The applicant of the present invention has found that the disruption of the blood-brain barrier in PD mice can lead to a large amount of plasma-derived fibrinogen (FG) entering the brain tissue, and the aggregation of FG in the brain can cause abnormal aggregation of α-syn and damage to dopaminergic neurons in the substantia nigra compacta. FG is a heterodimeric glycoprotein with a molecular weight of about 340 kDa, synthesized by hepatocytes, and mainly exists in plasma under normal physiological conditions, participating in the coagulation process of injury in blood vessels. In 2021, Silva et al. reported in the journal Science that the interaction between FG and cells mainly depends on integrin receptors. Integrin receptors are the main receptors for FG to bind to platelets, endothelial cells or macrophages in peripheral blood, and they are widely present in various cells, becoming an important bridge for the interaction between the extracellular matrix and cells. The applicant of the present invention further found that FG can promote the abnormal aggregation of α-syn and the increase of phosphorylated α-syn in neurons mediated by the neuronal αvβ3 integrin receptor, and cause mitochondrial damage to dopaminergic neurons, ultimately inducing the death of dopaminergic neurons. Inhibiting the binding of FG to the αvβ3 integrin receptor can effectively delay the pathological changes of PD.

[0004] GRGDSPLAPSC is a polypeptide containing arginine-glycine-aspartic acid (RGD). GRGDSPLAPSC is a competitive and reversible inhibitory peptide that can inhibit integrin-fibronectin binding. Currently, it has been found that GRGDSPLAPSC can be used to study the role of integrins in bone formation and resorption. No relevant research has been reported on using small molecule polypeptides related to the RGD module for the preparation of drugs for treating PD. Summary of the Invention

[0005] In order to overcome the above-mentioned disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide an application of a mimetic polypeptide containing a fibrinogen RGD module in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0006] The object of the present invention is achieved by the following solutions:

[0007] A mimetic polypeptide containing a fibrinogen RGD module, the sequence of the mimetic polypeptide is: Gly-Arg-Gly-Asp-Ser-Pro-Leu-Ala-Pro-Ser-Cys (GRGDSPLAPSC).

[0008] Among them, glycine (Gly, G), arginine (Arg, R), aspartic acid (Asp, D), serine (Ser, S), proline (Pro, P), leucine (Leu, L), alanine (Ala, A), proline (Pro, P), cysteine (Cys, C).

[0009] Furthermore, the protein molecular weight of the polypeptide is 1059.1.

[0010] The mimetic polypeptide of the present invention can be synthesized by the Fmoc solid-phase synthesis method, and the purity is identified by multi-effect liquid chromatography analysis.

[0011] Specifically, it may include the following steps:

[0012] 1. Resin swelling

[0013] Put the resin 2-Chlorotrityl Chloride Resin into a reaction tube, add DMF (15 mL / g), and shake for 60 min;

[0014] 2. Connect the first amino acid

[0015] Filter off the solvent through a sintered glass funnel, add 3-fold molar excess of Fmoc-protected amino acid (the first amino acid at the C-terminus), then add 10-fold molar excess of DIEA, and finally add DMF to dissolve, and shake for 30 min. Seal with methanol for 30 min.

[0016] 3. Deprotection

[0017] Remove DMF, add 20% piperidine DMF solution (15 mL / g) for 5 min, remove and then add 20% piperidine DMF solution (15 ml / g) for 15 min.

[0018] 4. Detection

[0019] Withdraw the piperidine solution. Take a dozen or so resin particles, wash them three times with ethanol, add 2 - 3 drops of Kaiser reagent, heat at 105°C - 110°C for 5 minutes, and a dark blue color indicates a positive reaction.

[0020] 5. Wash

[0021] Wash twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g).

[0022] 6. Condensation

[0023] Add 3 - fold molar excess of Fmoc - protected amino acid, 3 - fold molar excess of HBTU, then add 10 - fold molar excess of DIEA, and finally dissolve in DMF and shake for 45 minutes.

[0024] 7. Detection

[0025] Take a dozen or so resin particles, wash them three times with ethanol, add 2 - 3 drops of Kaiser reagent, heat at 105°C - 110°C for 5 minutes, and a colorless result indicates a negative reaction.

[0026] 8. Wash

[0027] Wash once with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g).

[0028] 9. Repeat the operations from 3 - 8, connect them in sequence from right to left until the Fmoc protecting group of the last amino acid is removed.

[0029] 10. Wash the resin and drain it according to the following method

[0030] Wash twice with DMF (10 mL / g), three times with DCM (10 mL / g), four times with methanol (10 mL / g), and drain for 10 minutes.

[0031] 11. Cleavage

[0032] Prepare the cleavage solution (10 mL / g): 95% TFA; 2% water; 2% EDT; 1% TIS. Cleavage time: 180 minutes.

[0033] 12. Blow - dry and wash

[0034] Blow - dry the cleavage solution as much as possible with nitrogen, precipitate with ether, centrifuge to remove the supernatant, wash the precipitate six times with ether, and then volatilize it at room temperature.

[0035] 13. Purification and preparation.

[0036] (1) Take a little of the crude product and dissolve it in H2O / ACN.

[0037] (2) Take a small amount of the sample and analyze it on an HPLC analyzer to determine the elution time corresponding to the target peak.

[0038] (3) Use a C18 reversed-phase chromatography preparation system: Wavelength: 220 nm; Flow Rate: 15 mL / min; Inj.Vol: 20 mL; Column Temp: 25 °C; Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in Acetonitrile; Collect the target peak solution.

[0039] (4) Take a little of the target peak solution with a 1.5 mL centrifuge tube for mass spectrometry confirmation and purity detection.

[0040] 14. Lyophilize the qualified target peak solution.

[0041] 15. Identification: Take a small amount of the finished polypeptide product respectively for molecular weight identification by MS and purity identification by HPLC analysis.

[0042] 16. Pack the powdery polypeptide in a sealed manner and store it at -20 °C.

[0043] The present invention also provides the use of the above-mentioned mimic polypeptide containing the fibrinogen RGD module in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0044] In the technical solution of the present invention, the drugs, which may be the same or different, respectively include a therapeutically effective amount of the mimic polypeptide containing the fibrinogen RGD module.

[0045] In the technical solution of the present invention, the drugs, which may be the same or different, respectively can be made into various pharmaceutical dosage forms by conventional methods, and these dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal tablets, granules, infusion, pills, elixirs, suspensions, medicinal wines, tinctures, drops and other oral dosage forms for oral administration and dosage forms for administration other than oral administration such as injections, like injections, etc.

[0046] In the technical solution of the present invention, the drugs, which may be the same or different, respectively may also contain one or more pharmaceutically acceptable carriers or excipients.

[0047] Furthermore, the carrier or excipient may include diluents, wetting agents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants, preservatives, etc.

[0048] The applicant of the present invention has found through research that FG abnormally accumulates in the substantia nigra pars compacta of PD mice, and the excessively aggregated FG in the brain may promote the abnormal aggregation of α-syn in dopaminergic neurons mediated by the αvβ3 integrin receptor. The mimetic polypeptide provided by the present invention can effectively inhibit the abnormal aggregation of α-syn and the death of dopaminergic neurons caused by the abnormally accumulated FG in the brain, improve the motor ability of PD mice modeled by MPTP, and provide new targets and ideas for the treatment of PD. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0050] Figure 1 It is the effect diagram of the reversal of the up-regulation of α-syn oligomer expression caused by FG intervention by the mimetic polypeptide of the present invention.

[0051] Figure 2 It is the effect diagram of the reduction of α-syn oligomer and phosphorylation levels in the substantia nigra of a PD mouse model modeled by MPTP by batroxobin.

[0052] Figure 3 It is the diagram of the reduction of pathological α-syn abnormal aggregation in SH-SY5Y cells induced by FG by an αvβ3 integrin receptor inhibitor.

[0053] Figure 4 It is the effect diagram of the reduction of the loss of dopaminergic neurons in PD mice modeled by MPTP by the mimetic polypeptide of the present invention.

[0054] Figure 5 It is the effect diagram of the reversal of the motor disorder in PD mice modeled by MPTP by the mimetic polypeptide of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0055] The present invention will be further described in detail below in conjunction with the embodiments, but the embodiments of the present invention are not limited thereto. The materials involved in the following embodiments can be obtained from commercial channels without special instructions. The methods are conventional methods without special instructions.

[0056] The mimic polypeptide of the present invention is screened for up to 20,000 polypeptide drugs or other known small molecule drugs in the existing polypeptide library according to the RGD peptide segment of FG through computer-aided drug design (CADD), and then a mimic polypeptide containing the RGD site of the fibrinogen (FG) α-chain is designed to match the αvβ3 integrin target. Finally, the optimal mimic peptide that can bind to this target is screened according to the binding energy parameter, and the polypeptide sequence is GRGDSPLAPSC.

[0057] The mimic polypeptide used in this example was synthesized by Qiangyao Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method, and the purity of the peptide reached over 95%. GRGDSPLAPSC can competitively inhibit the binding of FG to the neuronal αvβ3 integrin receptor by mimicking the RGD site in FG.

[0058] Reagent source: αvβ3 integrin receptor inhibitor (cyclo(RGDyK)): Supplier MCE, product number: HY-100563A; αvβ5 integrin receptor inhibitor (αvβ5integrin-IN-1): Supplier MCE, product number HY-145363; α5β1 integrin receptor inhibitor (ATN-161): Supplier MCE, product number HY-13535.

[0059] Example 1

[0060] Human neuroblastoma cells (SH-SY5Y cell line) expressing tyrosine hydroxylase (TH) were selected for in vitro culture. After resuscitation, the cells were replaced with high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% double-stranded antibiotics. When the cells were growing normally, the cell culture medium could be changed every 1-2 days. The growth and status of the cells needed to be observed every day, and attention should be paid to aseptic operation to avoid cell contamination.

[0061] When the cells grew to about 70%-90% covering the bottom surface, cell passage was carried out. The old cell culture medium was aspirated with a pipette tip, and an appropriate amount of sterile PBS buffer was added to wash the bottom surface of the culture flask. After sucking out the PBS buffer completely, as adherent cells, about 1 mL of trypsin digestion solution was added to SH-SY5Y for digestion at room temperature for 1-2 min. When most of the cells became round under the microscope, 1 mL of DMEM medium containing was added to terminate the digestion. One day before treatment with fibrinogen FG, the SH-SY5Y cells were counted with a cell counter. If the cells grew well and were free of contamination, the cell culture medium was changed, and each well of cells was replaced with DMEM culture medium without serum and antibiotics.

[0062] Then, 400 μg / mL of human serum-derived FG was added to the culture medium, and 400 μg / mL of GRGDSPLAPSC was added to the GRGDSPLAPSC treatment group. After adding, the culture solution was shaken well and incubated in the cell culture incubator for another 48 hours. The cell growth and morphology were observed every 12 hours to check if they were normal. After 48 hours, the cells were collected for experiments.

[0063] After the intervention time ended (48 hours), the cell culture medium was discarded, and the cells were washed 3 times with PBS buffer. Then, the cells were scraped off completely with a cell scraper. The scraped / blown cells were transferred to a 1.5 mL EP tube and centrifuged at 3000 r / min at 4°C for 10 minutes to retain the cell pellet. A certain amount of PBS buffer was added again and centrifuged at 3000 r / min at 4°C for 10 minutes. Subsequently, the pellet was retained, and a cell total protein lysis solution mixture was added. 100 - 200 μL of the prepared total protein lysis solution was added to each tube of cells, and they were repeatedly aspirated and shaken well, then left standing on ice for 30 - 60 minutes. Then, they were centrifuged at 12000 r / min at 4°C for 15 minutes, and the supernatant was taken into a new pre-cooled 1.5 mL EP tube. This was the prepared total protein sample, and then the total protein sample was used for Western Blot experiments to detect the protein expression level of total α-syn.

[0064] The experimental results showed that FG intervention would increase the expression of α-syn oligomers, but this trend could be significantly reversed after treating the cells with 400 μg / mL GRGDSPLAPSC, as Figure 1 shown.

[0065] Example 2

[0066] About 40 healthy adult male C57BL / 6 mice weighing about 25 g were randomly divided into four groups. One group of mice was injected with normal saline, and one group of mice was intraperitoneally injected with 30 mg / mL of 1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP) once a day for 5 consecutive days to establish a model. While establishing the model, a group of the MPTP group was injected with 30 BU / kg / d of batroxobin to promote the reduction of FG in whole blood (batroxobin was injected after injecting MPTP on the 5th day), and at the same time, a group of the normal saline group was injected with the same amount of batroxobin as a control. The mice were sacrificed 16 days after the model establishment, and the brain tissue of the substantia nigra compacta of the mice was ground and centrifuged to extract total protein for Western Blot experiments to detect the protein expression levels of total α-syn and phosphorylated α-syn.

[0067] The experimental results showed that after reducing the whole blood FG level with batroxobin, the pathological α-synuclein level in the substantia nigra of the PD mouse model established by MPTP was significantly reduced, suggesting that FG may play a role in promoting the abnormal aggregation of α-synuclein in PD mice. Reducing the level of FG can effectively reverse the aggregation process of α-synuclein, as Figure 2 shown.

[0068] Example 3

[0069] The SH-SY5Y cell line was passaged into 6-well plates, and 2 mL of DMEM culture medium was added to each well plate. The cells were randomly divided into 5 groups. The FG treatment group added 400 μg / mL of human serum-derived FG to the culture medium, the normal control group added an equal amount of PBS buffer, and the other three groups added corresponding concentrations of αvβ3 integrin receptor inhibitor, αvβ5 integrin receptor inhibitor, and α5β1 integrin receptor inhibitor according to the instructions on the basis of FG treatment. After 48 hours, the cells were collected, and total protein was extracted for Western Blot experiment to detect the protein expression level of total α-synuclein polymers. The experimental results suggested that the αvβ3 integrin receptor inhibitor significantly reduced the abnormal aggregation of pathological α-synuclein induced by FG, suggesting that FG promotes α-synuclein aggregation through the mediation of the αvβ3 integrin receptor, as Figure 3 shown.

[0070] Example 4

[0071] Forty healthy adult male C57BL / 6 mice weighing about 25 g were randomly divided into four groups. One group of mice was injected with normal saline, one group of mice was intraperitoneally injected with 30 mg / mL of 1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP) once a day for 5 consecutive days, one group of mice was subjected to MPTP modeling and GRGDSPLAPSC (5 days before MPTP modeling, GRGDSPLAPSC was diluted to 1.5 mg / mL with ACSF (artificial cerebrospinal fluid, commercially available routinely), and 2 μL was stereotaxically injected into the substantia nigra compacta), and at the same time, a group of normal saline group was injected with the same amount of GRGDSPLAPSC as a control. After 16 days of MPTP modeling, the mice were sacrificed, and the whole brain was fixed, dehydrated, and then coronal sections of the substantia nigra were cut. Immunohistochemistry was used to detect the number of dopaminergic neurons (tyrosine hydroxylase TH-positive neurons) in the substantia nigra of mice. The experimental results showed that GRGDSPLAPSC could significantly reduce the loss of dopaminergic neurons in PD mice modeled by MPTP, as Figure 4 shown.

[0072] Example 5

[0073] In Example 4, behavioral tests were performed on mice 3 days before they were killed, including (1) fatigue rotating rod test: mice were trained for 3 days before the experiment. The mice were placed on a fixed rod, and the rotation speed was gradually increased to 5 rpm to train them to rotate with the rod. Five training sessions were conducted at a speed of 5 rpm for 3 consecutive days (each training session was a maximum of 10 rotations), and the optimal state needed to be reached within 180 seconds. After the 3-day adaptation training, the test began, and the mice were placed in the center of a stationary drum with the body axis perpendicular to the drum axis. The rotation speed was gradually increased to 5 rpm, and the time the mice remained on the drum without falling was recorded (if it exceeded 180 seconds, it was recorded as 180 seconds). Each mouse was tested 3 times, each time with an interval of 30 minutes, and the average value was taken; (2) climbing pole test: the wooden pole was 0.6 cm wide and 50 cm high, with a rough surface. All mice were subjected to adaptation training 1 day before the experiment. During the experiment, the mouse was placed on the top of a wooden pole with its head facing upwards. The time it took to turn downwards and the time it took to crawl from the top of the pole to the ground (all four limbs must be completely on the ground) were recorded. The experiment was repeated for 5 consecutive times, and the best result was taken as the final result. Figure 5 From left to right in the figure, the total time of the pole climbing test, the turning time, and the time of the fatigue rotarod test are shown. The experimental results showed that GRGDSPLAPSC can significantly reverse the movement disorder of MPTP-induced PD mice.

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a mimetic polypeptide comprising a fibrinogen RGD module in the preparation of a drug for preventing and / or treating Parkinson's disease.

2. The use according to claim 1, characterized in that The sequence of the simulated polypeptide is: Gly-Arg-Gly-Asp-Ser-Pro-Leu-Ala-Pro-Ser-Cys.

3. The use according to claim 1, characterized in that: The drug comprises a therapeutically effective amount of a mimetic polypeptide comprising a fibrinogen RGD module.

4. The use according to claim 1, characterized in that: The medicine is prepared into various pharmaceutical dosage forms by conventional methods, and these dosage forms include: tablets, capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, oral dosage forms of drops and injections.

5. The use according to claim 1, characterized in that: The drug is prepared into various pharmaceutical dosage forms by conventional methods, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, oral dosage forms of drops and injections.

6. The use according to claim 1, characterized in that: The medicine also contains one or more pharmaceutically acceptable carriers or excipients.

7. The use according to claim 1, characterized in that: The carrier or auxiliary material includes at least one of a diluent, a wetting agent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant, and a preservative.

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