Use of a mimetic polypeptide comprising a fibrinogen RGD module in the preparation of a medicament for the prevention and / or treatment of Parkinson's disease

Abstract

The application belongs to the technical field of biological medicine, and discloses application of a simulation polypeptide containing a fibrinogen RGD module in preparation of a medicine for preventing and / or treating Parkinson's disease. The sequence of the simulation polypeptide containing the fibrinogen RGD module is Gly-Arg-Gly-Asp-Ser-Pro-Lys-Asn-Pro-Ser-Cys. The applicant of the application finds that FG abnormally accumulates in the substantia nigra compacta of a PD mouse, and the FG excessively accumulated in the brain may promote abnormal aggregation of alpha-syn of dopaminergic neurons through mediation of an alpha v beta 3 integrin receptor. The simulation polypeptide provided by the application can effectively inhibit abnormal aggregation of alpha-syn of dopaminergic neurons caused by the abnormally accumulated FG in the brain and death of the dopaminergic neurons, improve the motor ability of the PD mouse modeled by MPTP, and provide a new target and thought for PD treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of a mimic polypeptide containing a fibrinogen RGD module in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD), the second most common neurodegenerative disease, is characterized by the destruction of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies containing α-synuclein (α-syn). Currently, there is no effective cure or treatment for PD. Therefore, research into the pathological mechanisms of PD to identify more potential therapeutic targets and strategies is of great clinical significance.

[0003] The pathological mechanism of abnormal α-synuclein accumulation in Parkinson's disease (PD) is highly complex. Studies have shown that blood-brain barrier disruption in PD is closely related to abnormal α-synuclein accumulation. Our research has found that blood-brain barrier disruption in PD mice leads to the entry of large amounts of plasma-derived fibrinogen (FG) into brain tissue. The accumulation of FG in the brain can cause abnormal α-synuclein accumulation and damage to dopaminergic neurons in the substantia nigra pars compacta. FG is a heterodimeric glycoprotein with a molecular weight of approximately 340 kDa, synthesized by hepatocytes. Under normal physiological conditions, it is mainly present in plasma and participates in the coagulation process in blood vessels. In 2021, Silva et al. reported in Science that the interaction between FG and cells mainly depends on integrin receptors. Integrin receptors are the main receptors for FG binding to platelets, endothelial cells, and macrophages in peripheral blood. They are widely present in various cell types, serving as an important bridge for extracellular matrix-cell interaction. The applicant of this invention further discovered that FG can promote the abnormal accumulation of α-syn and the increase of phosphorylated α-syn through neuronal αvβ3 integrin receptor, leading to mitochondrial damage in dopaminergic neurons and ultimately inducing the death of dopaminergic neurons. Inhibiting the binding of FG to 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 inhibits integrin-fibronectin binding. Current research suggests that GRGDSPLAPSC can be used to study the role of integrin in bone formation and resorption. No studies have yet utilized small molecule polypeptides related to the RGD module for the preparation of drugs to treat Parkinson's disease (PD). Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide the application of a mimicry polypeptide containing a fibrinogen RGD module in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0006] The objective of this invention is achieved through the following solution:

[0007] A mimic polypeptide comprising a fibrinogen RGD module, the sequence of which 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), and cysteine ​​(Cys, C).

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

[0010] The simulated polypeptide described in this invention can be synthesized by the Fmoc solid-phase synthesis method, and its purity can be identified by multi-effect liquid chromatography analysis.

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

[0012] 1. Resin swelling

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

[0014] 2. Receive the first amino acid.

[0015] The solvent was removed by filtration through a sand filter. A 3-molar excess of Fmoc was added to protect the amino acid (the first amino acid at the C-terminus), followed by a 10-molar excess of DIEA. Finally, DMF was added to dissolve the mixture, and the mixture was shaken for 30 minutes. The mixture was then capped with methanol and incubated for 30 minutes.

[0016] 3. Deprotection

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

[0018] 4. Testing

[0019] Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add 2-3 drops of Kaiser's reagent, heat at 105℃-110℃ for 5 minutes, and a deep blue color indicates a positive reaction.

[0020] 5. Wash

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

[0022] 6. Condensation

[0023] Add 3 times molar excess of Fmoc to protect amino acids, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, and finally add DMF to dissolve. Shake for 45 minutes.

[0024] 7. Testing

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

[0026] 8. Wash

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

[0028] 9. Repeat steps 3-8, connecting from right to left, until the last amino acid Fmoc protecting group is removed.

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

[0030] DMF (10 mL / g) twice, DCM (10 mL / g) three times, methanol (10 mL / g) four times, and then the mixture was dried under vacuum for 10 minutes.

[0031] 11. Cutting

[0032] Prepare the cutting fluid (10 mL / g): TFA 95%; water 2%; EDT 2%; TIS 1%. Cutting time: 180 min.

[0033] 12. Dry and wash

[0034] The lysis buffer was dried as much as possible with nitrogen gas, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed six times with ether, and then evaporated to dryness at room temperature.

[0035] 13. Purification and preparation.

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

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

[0038] (3) Using a C18 reversed-phase chromatography 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 small amount of the target peak solution in a 1.5 mL centrifuge tube for mass spectrometry confirmation and purity detection.

[0040] 14. Freeze-dry the qualified target peak solution.

[0041] 15. Identification: Take small amounts of the finished polypeptide and perform molecular weight identification by MS and purity identification by HPLC.

[0042] 16. Seal the powdered peptides and store them 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 the prevention and / or treatment of Parkinson's disease.

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

[0045] In the technical solution of this invention, the same or different drugs can be made into various pharmaceutical dosage forms using conventional methods. These dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, elixirs, suspensions, tinctures, drops, and other oral dosage forms, as well as non-oral dosage forms such as injections.

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

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

[0048] The applicant of this invention discovered that fibroblasts (FG) abnormally accumulate in the substantia nigra pars compacta of PD mice, and that excessive FG accumulation in the brain may promote abnormal α-synuclein aggregation in dopaminergic neurons through αvβ3 integrin receptor mediation. The mimic peptide provided by this invention can effectively inhibit the abnormal α-synuclein aggregation and dopaminergic neuronal death caused by abnormally accumulated FG in the brain, improve the motor function of PD mice induced by MPTP, and provide new targets and strategies for PD treatment. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used 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 should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is a diagram showing the effect of the present invention in reversing the upregulation of α-syn multimer expression induced by FG ​​intervention with simulated peptides.

[0051] Figure 2 The effect of batroxobin on reducing MPTP levels and phosphorylation in a PD mouse model.

[0052] Figure 3 A diagram showing how an αvβ3 integrin receptor inhibitor reduces FG-induced pathological α-syn abnormal aggregation in SH-SY5Y cells.

[0053] Figure 4 This is a diagram showing the effect of the simulated peptide of the present invention in reducing the loss of dopaminergic neurons in PD mice modeled with MPTP.

[0054] Figure 5 This is a diagram showing the effect of the present invention on motor dysfunction in PD mice modeled by the reversal of MPTP by a polypeptide. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0056] The mimic peptide of this invention utilizes computer-aided drug design (CADD) to screen up to 20,000 peptide drugs or other known small molecule drugs from an existing peptide library based on the RGD peptide of fibrinogen (FG). Then, it designs a mimic peptide containing the RGD site of the fibrinogen (FG) α chain to match the αvβ3 integrin target. Finally, it screens the optimal mimic peptide that can bind to the target based on the binding energy parameter. The peptide sequence is GRGDSPLAPSC.

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

[0058] Reagent sources: αvβ3 integrin receptor inhibitor (cyclo(RGDyK)): MCE product number: HY-100563A; αvβ5 integrin receptor inhibitor (αvβ5integrin-IN-1): MCE product number: HY-145363; α5β1 integrin receptor inhibitor (ATN-161): 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. Under normal cell growth conditions, the cell medium could be changed every 1-2 days. Cell growth and status should be observed daily, and aseptic operation should be observed to avoid cell contamination.

[0061] When the cells reach approximately 70%-90% coverage of the bottom surface, passage them. Aspirate the old culture medium with a pipette tip and add an appropriate amount of sterile PBS buffer to wash the bottom of the culture flask. After removing the PBS buffer, for SH-SY5Y cells (adherent cells), passage requires adding approximately 1 mL of trypsin digest and digesting at room temperature for 1-2 minutes. When most cells are observed to be rounded under a microscope, add 1 mL of DMEM medium to stop the digestion. The day before fibrinogen (FG) treatment, count the SH-SY5Y cells using a cell counter. If the cells are growing well and are free of contamination, change the medium, replacing each well with serum- and antibiotic-free DMEM medium.

[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 medium was shaken thoroughly and incubated in a cell culture incubator for 48 hours. Cell growth and morphology were observed every 12 hours. Cells were collected after 48 hours for experiments.

[0063] After the intervention period (48 hours), the cell culture medium was discarded, and the cells were washed three times with PBS buffer. All cells were then scraped off using a cell scraper and transferred to 1.5 mL EP tubes. The cells were centrifuged at 4°C and 3000 rpm for 10 min, and the cell pellet was retained. A certain amount of PBS buffer was added, and the cells were centrifuged again at 4°C and 3000 rpm for 10 min. The pellet was then retained, and a total protein lysis buffer mixture was added. 100-200 μL of the prepared total protein lysis buffer was added to each cell tube, and the mixture was repeatedly aspirated and vortexed to mix. The mixture was then placed on ice for 30-60 min. Finally, the cells were centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was collected into a new pre-chilled 1.5 mL EP tube. This was the extracted total protein sample. Western blotting was then performed on the total protein sample to detect the total α-syn protein expression level.

[0064] Experimental results showed that FG intervention increased α-synuclein multimer expression, but treatment with 400 μg / mL GRGDSPLAPSC significantly reversed this trend. Figure 1 As shown.

[0065] Example 2

[0066] Forty healthy adult male C57BL / 6 mice (approximately 25g each) were randomly divided into four groups. One group was injected with saline, while the other group underwent intraperitoneal injection of 30mg / mL 1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP) once daily for 5 consecutive days to induce a model. Simultaneously, the MPTP group was injected with 30BU / kg / d batroxobin to promote a reduction in whole blood free radicals (FG) (batroxobin was injected after MPTP injection on day 5). The saline group was injected with the same amount of batroxobin as a control. Sixteen days after the model was established, the mice were sacrificed, and the substantia nigra pars compacta brain tissue was collected, ground, centrifuged, and total protein was extracted for Western blotting to detect the protein expression levels of total α-syn and phosphorylated α-syn.

[0067] Experimental results showed that batroxobin significantly reduced the level of pathological α-synuclein protein in the substantia nigra of MPTP-induced PD mouse models after reducing whole blood FG levels, suggesting that FG may promote abnormal α-synuclein aggregation in PD mice, and reducing FG levels can effectively reverse the α-synuclein aggregation process. Figure 2 As shown.

[0068] Example 3

[0069] SH-SY5Y cell lines were passaged into 6-well plates, with 2 mL of LDM medium added to each well. Cells were randomly divided into 5 groups: the FG treatment group received 400 μg / mL human serum-derived FG in the medium; the normal control group received an equal volume of PBS buffer; and the other three groups received FG treatment plus the corresponding concentrations of αvβ3 integrin receptor inhibitors, αvβ5 integrin receptor inhibitors, and α5β1 integrin receptor inhibitors, respectively, according to the manufacturer's instructions. Cells were collected after 48 hours, and total protein was extracted for Western blotting to detect the protein expression level of total α-synuclein multimers. The results showed that αvβ3 integrin receptor inhibitors significantly reduced FG-induced pathological α-synuclein aggregation, suggesting that FG promotes α-synuclein aggregation through αvβ3 integrin receptor mediation. Figure 3 As shown.

[0070] Example 4

[0071] Forty healthy adult male C57BL / 6 mice (approximately 25g each) were randomly divided into four groups. One group received saline injections. Another group underwent intraperitoneal injection of 30mg / mL 1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP) once daily for 5 consecutive days to establish a PD model. A third group underwent MPTP modeling and received GRGDSPLAPSC (5 days before MPTP modeling, GRGDSPLAPSC was diluted to 1.5mg / mL with ACSF (commercially available artificial cerebrospinal fluid) and injected stereotactically into the substantia nigra pars compacta, using 2μL). Simultaneously, the saline group received the same amount of GRGDSPLAPSC as a control. Sixteen days after MPTP modeling, the mice were sacrificed, and the entire brain was fixed, dehydrated, and sliced ​​from the substantia nigra. Immunohistochemical staining was performed to detect the number of dopaminergic neurons (tyrosine hydroxylase TH-positive neurons) in the substantia nigra. The results showed that GRGDSPLAPSC significantly reduced the loss of dopaminergic neurons in MPTP-modeled PD mice. Figure 4 As shown.

[0072] Example 5

[0073] In Example 4, behavioral tests were conducted on the mice 3 days before their sacrifice, including (1) fatigue rotundus test: The 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. At a speed of 5 rpm, the mice were trained 5 times a day for 3 consecutive days (each training session was limited to a maximum of 10 rotations) and needed to reach the optimal state within 180s. After the 3-day adaptation training, the test began. The mice were placed in the center of a stationary roller with their body axis perpendicular to the roller axis. The rotation speed was gradually increased to 5 rpm, and the time it took for the mice to remain on the roller without falling was recorded (if it exceeded 180s, it was recorded as 180s). Each mouse was tested 3 times, with an interval of 30 minutes between each test, and the average value was taken; (2) pole climbing test: The wooden pole was 0.6 cm wide and 50 cm high with a rough surface. All mice were given adaptation training 1 day before the experiment. In the experiment, mice were placed on top of a wooden pole with their heads facing upwards. The time it took for them to turn around and descend, as well as the time it took for them to climb from the top of the pole to the ground (all four limbs must be fully on the ground), was recorded. This was repeated five times, and the best result was used as the final result. See [link to results]. Figure 5 The figure, from left to right, shows the total time for the pole climbing experiment, the turning time, and the time for the fatigue twirling experiment. Experimental results show that GRGDSPLAPSC can significantly reverse motor impairment in MPTP-modeled PD mice.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a mimic polypeptide containing a fibrinogen RGD module in the preparation of a drug for treating Parkinson's disease, characterized in that... The sequence of the simulated polypeptide is: Gly-Arg-Gly-Asp-Ser-Pro-Leu-Ala-Pro-Ser-Cys.

2. The application according to claim 1, characterized in that: The drug comprises a therapeutically effective amount of a mimic polypeptide containing a fibrinogen RGD module.

3. The application according to claim 1, characterized in that: The drug is formulated into various pharmaceutical dosage forms, including: tablets, capsules, oral liquids, lozenges, granules, pills, elixirs, suspensions, tinctures, and injections for oral administration.

4. The application according to claim 1, characterized in that: The drug is formulated into various pharmaceutical dosage forms, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, and soft capsules.

5. The application according to claim 1, characterized in that: The drug also contains one or more pharmaceutically acceptable carriers or excipients.

6. The application according to claim 5, characterized in that: The carrier or excipient includes at least one of diluent, wetting agent, adhesive, surfactant, adsorbent carrier, lubricant, disintegrant, and preservative.

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