Use of 1-allylpiperazine for the preparation of a medicament for the prevention and treatment of Parkinson's disease

Abstract

The present application relates to the technical field of biological medicine, in particular to the application of 1-allyl piperazine in the preparation of drugs for preventing and treating Parkinson's disease. The present application discloses for the first time that 1-allyl piperazine can significantly inhibit the aggregation of alpha-synuclein in vitro. 1-allyl piperazine can significantly reduce the cytotoxicity induced by alpha-synuclein aggregation, and 1-allyl piperazine can effectively interfere with the liquid-liquid phase separation process of alpha-synuclein. It is also found through computer simulation data that 1-allyl piperazine can interact with the target (alpha-synuclein). It is proved that the 1-allyl piperazine has an anti-Parkinson's disease effect and can be applied to the preparation of Parkinson's disease prevention and treatment preparations.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of 1-allylpiperazine in the preparation of drugs for the prevention and treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common chronic, disabling neurodegenerative disease affecting the elderly, characterized by bradykinesia, resting tremor, and rigidity. PD predominantly affects the elderly, placing a significant burden on patients and their families. Current interventions can only temporarily alleviate some symptoms; they cannot effectively slow or halt the disease's progression, nor can they achieve a complete cure.

[0003] The hallmark pathological features of Parkinson's disease are the specific loss of dopaminergic (DA) neurons in the substantia nigra of the midbrain and the presence of Lewy bodies (LBs). α-synuclein, a major structural component of Lewy bodies, exists as a soluble monomer under physiological conditions. However, under pathological conditions, this protein misfolds and abnormally aggregates, forming neurotoxic oligomers and insoluble fibers, ultimately maturing into Lewy bodies.

[0004] There are no existing reports on the prevention and treatment of Parkinson's disease with 1-allylpiperazine. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of 1-allylpiperazine in the preparation of drugs for the prevention and treatment of Parkinson's disease, wherein 1-allylpiperazine can prevent and treat Parkinson's disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of 1-allylpiperazine in the preparation of drugs for the prevention and treatment of Parkinson's disease.

[0008] Preferably, the 1-allylpiperazine prevents and treats Parkinson's disease by inhibiting the aggregation of α-synuclein.

[0009] Preferably, the 1-allylpiperazine inhibits the aggregation of α-synuclein by binding to α-synuclein, disrupting the β-sheet structure, and reducing stability.

[0010] Preferably, the 1-allylpiperazine prevents and treats Parkinson's disease by antagonizing α-synuclein-induced cytotoxicity.

[0011] Preferably, the 1-allylpiperazine prevents and treats Parkinson's disease by interfering with the liquid-liquid phase separation of α-synuclein.

[0012] Preferably, the concentration of the 1-allylpiperazine used is above 25 µM.

[0013] Preferably, the dosage form of the drug includes one or more of oral preparations, injectable preparations, powders, granules, powders, and tablets.

[0014] The present invention also provides a drug for the prevention and treatment of Parkinson's disease, comprising 1-allylpiperazine.

[0015] Preferably, pharmaceutically acceptable carriers are also included.

[0016] Preferably, the dosage form of the drug includes one or more of oral preparations, injectable preparations, powders, granules, powders, and tablets.

[0017] The beneficial effects of this invention are:

[0018] This invention discloses for the first time that 1-allylpiperazine can significantly inhibit the aggregation of α-synuclein in vitro. 1-Allylpiperazine can significantly alleviate the cytotoxicity induced by α-synuclein aggregation and effectively interfere with the liquid-liquid phase separation process of α-synuclein. Computer simulations also revealed that 1-allylpiperazine can interact with its target (α-synuclein). This demonstrates that 1-allylpiperazine possesses anti-Parkinson's disease activity and can be applied to the preparation of agents for the prevention and treatment of Parkinson's disease. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 The graph shows the change in relative ThT fluorescence intensity with incubation time in the presence or absence of α-Syn.

[0021] Figure 2 The graph shows the ThT fluorescence intensity at different concentrations of 1-ALPP.

[0022] Figure 3 The graph shows the ThT fluorescence intensity over 10 days in the presence of different concentrations of 1-ALPP.

[0023] Figure 4 A statistical graph showing the effect of different concentrations of 1-allylpiperazine on the survival rate of PC12 cells;

[0024] Figure 5 Statistical graph showing the effect of different ratios of 1-allylpiperazine on inhibiting α-Syn on PC12 cell survival;

[0025] Figure 6To investigate the effect of 1-ALPP on liquid-liquid phase separation, FITC-labeled α-synuclein (green fluorescence) at a concentration of 30 µM was incubated in a buffer solution, and the samples were divided into control group (no 1-ALPP) and treatment group (1-ALPP). The samples were observed under a fluorescence microscope immediately after preparation, with an excitation wavelength of 546 nm. Scale bar: 50 µm.

[0026] Figure 7 Particle size variation in LLPS system

[0027] Figure 8 This represents the radial distribution function between 1-ALPP and α-synuclein;

[0028] Figure 9 The number of interchain contacts of α-synuclein in the presence and absence of 1-ALPP;

[0029] Figure 10 (A) A typical snapshot of the α-synuclein pentamer during a 100 ns molecular dynamics simulation in a pure α-synuclein system; (B) A typical snapshot obtained during a 100 ns molecular dynamics simulation in a 1-ALPP-α-synuclein complex system.

[0030] Figure 11 The evolution of the secondary structure of the α-synuclein pentamer in the presence of (A) and the absence of (B) 1-ALPP;

[0031] Figure 12 Effects of 1-ALPP on α-synuclein aggregation in PD model nematode NL5901 (A: control, B: nematodes fed with 75 μM 1-ALPP).

[0032] Among them, control is the control group. Detailed Implementation

[0033] This invention provides the application of 1-allylpiperazine in the preparation of drugs for the prevention and treatment of Parkinson's disease.

[0034] 1-Allylpiperazine (1-ALPP) was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (brand: Leyan, product catalog number: 13961-36-9), and its structural formula is as follows:

[0035] In this invention, the 1-allylpiperazine preferably prevents and treats Parkinson's disease by inhibiting the aggregation of α-synuclein. In this invention, the 1-allylpiperazine preferably inhibits the aggregation of α-synuclein by binding to it, disrupting its β-sheet structure, and reducing its stability. In this invention, the 1-allylpiperazine preferably prevents and treats Parkinson's disease by antagonizing α-synuclein-induced cytotoxicity. In this invention, the 1-allylpiperazine preferably prevents and treats Parkinson's disease by interfering with the liquid-liquid phase separation of α-synuclein. In this invention, the concentration of the 1-allylpiperazine used is preferably 25 µM or higher. In this invention, the dosage form of the drug preferably includes one or more of oral formulations, injectable formulations, powders, granules, tablets, and other similar formulations.

[0036] This invention provides a medicament for the prevention and treatment of Parkinson's disease, comprising 1-allylpiperazine. In this invention, the medicament preferably also comprises a pharmaceutically acceptable carrier. The dosage form of the medicament includes one or more of the following: oral formulations, injectable formulations, powders, granules, tablets, and granules.

[0037] To further illustrate the present invention, the following detailed description of the embodiments is provided, but these should not be construed as limiting the scope of protection of the present invention. Example 1

[0038] Changes in ThT fluorescence intensity over time when different concentrations of 1-allylpiperazine were co-incubated with α-Syn

[0039] Experimental Procedure: Using the *E. coli* strain that previously constructed the recombinant α-syn protein (paper title: High-efficiency expression, purification, and characterization of α-synuclein DOI: 10.13364 / j.issn.1672-6510.20180348), recombinant α-synuclein was expressed in *E. coli* BL21 cells under shaking conditions at 37°C. When the cell optical density reached 0.6 to 0.8, 0.5 mM IPTG was added to induce expression, and the cells were cultured at 37°C for another 4 hours. After expression, the cells were collected, and the target protein was purified using nickel affinity chromatography. Finally, the obtained recombinant protein was lyophilized and stored at -80°C for later use.

[0040] The lyophilized α-syn powder was reconstituted in 20 mM Tris-HCl buffer (TBS, pH 7.4) to prepare a working solution with a final concentration of 50 μM. To remove pre-existing aggregates, the reconstituted solution was sonicated at 4°C for 15 min, followed by centrifugation at 14,000 × g for 15 min at 4°C. The supernatant was used as the monomeric α-syn stock solution. The inhibitor 1-ALPP (10 mM) was prepared using the same 20 mM TBS buffer.

[0041] The fibrillation kinetics of α-syn were monitored using ThT fluorescence. In 96-well black opaque plates, 200 μL of α-syn monomer solution (50 μM) was mixed with 20 μL of ThT solution (250 μM) to achieve final α-syn and ThT concentrations of 45.5 μM and 22.7 μM, respectively. To investigate the inhibitory effect of 1-ALPP, different volumes of 1-ALPP stock solution were added to the reaction systems of some experimental groups, with an equal volume of TBS buffer as a control. After sealing the plates with a sealing film, they were placed in a microplate reader (TECAN Infinite 200PRO, Austria) and incubated at 37°C while monitoring fluorescence signals. The fluorescence reading conditions were set as follows: excitation wavelength 440 nm and emission wavelength 480 nm.

[0042] Experimental results: such as Figure 1 As shown, within the experimental concentration range, 1-ALPP itself produces almost no ThT fluorescence signal, indicating that its presence does not interfere with ThT fluorescence detection. Figure 2 As shown, when 50 μM α-syn was incubated alone, its ThT fluorescence kinetic curve exhibited typical characteristics of amyloid nucleation and aggregation. When 50 μM α-syn was co-incubated with different concentrations of 1-ALPP, 1-ALPP showed a concentration-dependent inhibitory effect: under the conditions of 1-ALPP to α-syn molar ratios of 0.5:1, 1:1, and 2:1, after 10 days (e.g., ... Figure 3 The final fluorescence intensity of the system decreased sequentially to approximately 48.81%, 34.30%, and 16.45% of that of the control group. These results fully demonstrate that 1-ALPP can effectively inhibit the fibrosis process of α-synuclein in a concentration-dependent manner. Example 2

[0043] CCK-8 assay for cytotoxicity

[0044] Implementation steps: The cells used in the cytotoxicity experiment were the mouse adrenal pheochromocytoma cell line (PC12).

[0045] Cells were cultured routinely in a 37°C, 5% CO2 incubator using RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged every 2-3 days, and all experiments used cells in the logarithmic growth phase. Before cytotoxicity assays, cells were counted in the logarithmic growth phase and then cultured at approximately 1 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 100 μL / well in 96-well plates. RPMI 1640 medium was added to each well to a total volume of 100 μL. Cells adhered after 24 hours. Experimental groups were treated with different concentrations of 1-ALPP, while the control group received an equal volume of PBS. Incubation continued for another 24 hours. After incubation, 10 μL of CCK-8 solution was slowly added to each well (avoiding air bubbles), and the plates were incubated at 37°C for 2 hours. The absorbance was then measured at 450 nm using a TECAN Infinite 200 PRO (Austria) microplate reader.

[0046] Experimental results: such as Figure 4 The concentrations of 1-ALPP used did not show significant cytotoxicity (Figure 4). Cytotoxicity was assessed after co-incubating different concentrations of 1-ALPP with α-syn for 11 days, and the results showed (…). Figure 5 Pure α-synuclein aggregates exhibited extremely strong cytotoxicity, reducing the viability of PC12 cells treated with them to 49.50%. 1-ALPP showed a significant protective effect against α-synuclein aggregation-induced cytotoxicity, and this effect was clearly concentration-dependent. Figure 5 As shown, treatment with 25 μM, 50 μM, and 100 μM 1-ALPP restored cell viability affected by α-synuclein aggregates to 65.78%, 82.47%, and 91.99%, respectively. This recovery effect across concentration gradients confirms that 1-ALPP can effectively antagonize the neurotoxic effects of α-synuclein. Example 3

[0047] In vitro fluorescein isothiocyanate labeling of target proteins

[0048] Implementation steps: Fluorescent labeling technology achieves the tracking and visualization of target proteins through the specific binding or covalent coupling of fluorescent dyes with target molecules. In this embodiment, fluorescein isothiocyanate (FITC, purchased from Beyotime, Shanghai, China) is selected as the labeling dye, and its covalent binding reaction with lysine residues is used to fluorescently label the target protein.

[0049] The labeling procedure is as follows: Dissolve 12.5 mg of FITC in 1 mL of DMSO to prepare a 12.5 mg / mL (approximately 32 mM) FITC stock solution. Mix the target protein with an appropriate amount of the FITC stock solution in 1 mL of 0.2 M NaHCO3 buffer (pH ~9.0). This alkaline environment is conducive to the reaction between FITC and protein amino groups. Make up the total volume of the reaction system to 2 mL with double-distilled water. Transfer the reaction solution to a 2 mL centrifuge tube, wrap it in aluminum foil to protect it from light, and incubate it on a shaker at room temperature (~200 rpm) for 2 hours. After the reaction, completely remove unbound free FITC by dialysis (molecular weight cutoff 8-14 kDa). For phase separation experiments, add approximately 10% of the volume of the labeled protein solution to the unlabeled protein stock solution, so that the labeling ratio is approximately 1%.

[0050] In vitro liquid-liquid phase separation experiment

[0051] Procedure: The liquid-liquid phase separation behavior of α-synuclein under different conditions was observed using a fluorescence microscope (Olympus, BX53, Japan). The phase separation reaction system consisted of the following components: 50 mM MES-Tris buffer (pH 6.0), 250 mM NaCl, 10% PEG8000, the target protein, and different concentrations of the test drug. All samples were freshly prepared. 5-10 μL was dropped onto a clean glass slide, a coverslip was gently placed on top (avoiding air bubbles), and the slide was immediately placed on the microscope stage for observation.

[0052] Given that the microflow of the liquid under the coverslip may affect image quality, a fast scanning mode was employed to ensure image sharpness and data repeatability. The imaging parameters were set as follows: excitation wavelength of 488 nm (for detecting FITC-labeled proteins), laser intensity of 50%, scan speed of 4 μs / pixel, and image resolution of 1024 × 1024 pixels. Using this imaging system, the effects of different drug concentrations on the morphology, size distribution, and number density of α-Syn phase-separated droplets were evaluated.

[0053] Experimental Results: Liquid-liquid phase separation (LLPS) is the initiating step in the pathological aggregation of α-synuclein (α-Syn). This process is driven by multivalent weak interactions and precisely regulated by microenvironmental factors, and is closely related to protein misfolding and amyloid fibrillation. To investigate the effect of compound 1-ALPP on the α-Syn LLPS process, this study used FITC-labeled α-Syn (30 µM) in a buffer system to observe its phase separation behavior. Fluorescence microscopy observations showed ( Figure 6In the control group, a large number of densely distributed α-Syn droplets exhibited typical LLPS characteristics. After the addition of 1-ALPP, the droplet morphology became more regular and clear. When the molar ratio of 1-ALPP to α-Syn was 1:1, the droplet size was smaller than that of the control group; although droplets of similar size to those in the control group were visible in the field of view, their number was significantly reduced. When the molar ratio increased to 3:1, both the size and number of droplets further decreased, exhibiting a dose-dependent inhibitory effect. These results indicate that 1-ALPP can effectively intervene in the LLPS process of α-Syn, and its effect may be achieved by interfering with the multivalent weak interaction network between α-Syn molecules, thereby blocking the normal growth and fusion of droplets. Example 4

[0054] The phase separation reaction system solution prepared in Example 3 above was filtered through a 0.22 µm filter membrane to remove large-sized impurities and air bubbles. Approximately 1 mL of filtrate was collected and transferred to a particle size measurement cuvette. Particle size was measured directly using a nanoparticle size analyzer (Zetasizer Pro, Malvern Panaco, UK). Each sample was measured 3-5 times, and the average value was used for subsequent data analysis.

[0055] Experimental Results: Due to the complexity of the LLPS in vitro system, fluorescence microscopy alone is insufficient to quantitatively distinguish the differences between different treatment groups. Therefore, this study used dynamic light scattering (DLS) to quantitatively characterize the particle size distribution of LLPS droplets. The results showed ( Figure 7 The particle sizes of all three groups of samples were concentrated in the logarithmic coordinate range of 100–1000 nm (submicron to micron scale), with no obvious signal in the 10–100 nm range, indicating the absence of interference from small-sized impurities. The Control group had the largest droplet size among the three groups, with a characteristic peak at approximately 900 nm and a peak intensity of approximately 22. When the molar ratio of 1-ALPP to α-Syn was 1:1, the characteristic peak shifted to approximately 660 nm, and the peak intensity decreased to approximately 18, with both droplet size and relative content significantly lower than the Control group. When the molar ratio increased to 3:1, the characteristic peak further shifted to approximately 300 nm, and the peak intensity decreased to approximately 15, making it the group with the smallest particle size and lowest relative content among the three groups. These results indicate that with increasing 1-ALPP concentration, the characteristic particle size of LLPS droplets exhibits a gradient decreasing trend, and the relative content of droplets decreases simultaneously. Compared with the Control group, both experimental groups showed a significant decrease in droplet size and content, confirming that 1-ALPP can effectively inhibit the nucleation and growth process of LLPS droplets through proportional control. Example 5

[0056] Molecular dynamics simulation study of the interaction between 1-allylpiperazine and α-synuclein

[0057] Implementation Steps: To reveal the interaction mode between the compounds of this invention and the target protein at the atomic level, molecular dynamics simulations were employed. The three-dimensional structure of the α-synuclein pentamer was obtained from the Protein Data Bank (PDBID: 6H6B). The structure of 1-allylpiperazine was obtained from the PubChem database (CAS: 13961-36-9). Using GROMACS2022.6 software and the Amber14SB force field, molecular dynamics simulations were performed for 100 ns on both the standalone α-synuclein pentamer system and the complex system containing 1-allylpiperazine. By calculating parameters such as binding free energy (MM / PBSA), root mean square deviation (RMSD), root mean square fluctuation (RMSF), and secondary structure evolution, the binding mode between the compounds and the target protein and their impact on protein structural stability were evaluated.

[0058] Experimental results: Simulation results indicate that 1-allylpiperazine forms a stable complex with α-synuclein. During the simulation process ( Figure 8-9 1-Allylpiperazine is primarily localized within a 1 nm radius around α-synuclein. In the presence of 1-ALPP, the number of contacts between the AB and DE chains of α-synuclein is significantly reduced, indicating that 1-ALPP loosens the structure of α-synuclein, thereby hindering its aggregation. MM / PBSA calculations (Table 1) show that the binding free energy is -30.32 kcal / mol, indicating strong binding affinity. In the simulated trajectory ( Figure 10 In the α-synuclein pentamer, the overall structure was maintained, with the β-sheet remaining the dominant secondary structure. In contrast, in the 1-ALPP-α-synuclein system, 1-ALPP gradually approached the α-synuclein within the first 50 ns and then bound stably within the next 50 ns, resulting in a looser structure and a reduced β-sheet content. RMSD, SASA, and RMSF results (Table 2) further confirmed that 1-ALPP disrupted the structural stability of the α-synuclein. Figure 11 The secondary structure evolution of the 1-ALPP-α-synuclein system and the α-synuclein system alone is shown. Under the influence of 1-ALPP, large areas of β-sheet regions—especially residues 10-40, 60-70, 120-140, 200, and 260-270—become random coils, thereby destabilizing the α-synuclein structure and ultimately reducing its aggregation tendency.

[0059] Table 1. Binding energy in the last 10 ns of simulation

[0060] Table 2. Root mean square deviation, root mean square fluctuation, and solvent-accessible surface area of ​​the α-synuclein system and the 1-ALPP-α-synuclein complex system during the simulation.

[0061] Molecular dynamics simulations revealed that 1-ALPP forms a stable complex with α-synuclein, with a binding energy of -30.32 kcal / mol. During the simulations, 1-ALPP gradually approaches and binds to α-synuclein, inducing several key regions to transition from β-sheets to random coils, significantly disrupting the protein's secondary structure. Furthermore, 1-ALPP reduces interchain contacts, decreasing the structural stability and compactness of α-synuclein, making the overall assembly more flexible, thereby effectively inhibiting α-synuclein aggregation. Example 6

[0062] Effects of different concentrations of 1-ALPP on Aβ42 aggregation in the AD model nematode NL5901.

[0063] L1-stage nematodes were flushed off the culture medium with M9 buffer and transferred to 15 mL centrifuge tubes. The supernatant was discarded after centrifugation. The nematodes were then transferred to NGM medium coated with E. coli OP50 and cultured at 20°C until the L4 stage. Synchronized nematodes were transferred to NGM medium. The blank control group consisted of NGM medium coated with OP50 and containing an equal volume of double-distilled water (equal to 1-ALPP). The final drug concentration in the experimental groups was 75 μM, with three replicates per group. Fifty nematodes were picked from each group and anesthetized with 4% NaN3. The aggregation of green fluorescent protein in the nematodes was observed using a laser confocal microscope. The fluorescence intensity of all images was quantitatively analyzed using ImageJ software. The results are as follows: Figure 12 As shown.

[0064] Experimental results: Figure 12 The results showed that the intensity of yellow-green fluorescence in nematodes fed with 75 μM 1-ALPP was weaker than that in the control group. Therefore, fluorescence intensity represents the amount of α-syn aggregation, and the lower the value, the stronger the anti-α-syn aggregation ability of 1-ALPP.

[0065] In summary, molecular dynamics simulations at the atomic level confirm that 1-allylpiperazine can inhibit the aggregation tendency of α-synuclein at the molecular level by directly binding to it, disrupting its key β-sheet structure, and reducing its structural stability. This mechanism is corroborated by in vitro experiments, demonstrating that the compound of this invention exerts its therapeutic effect on Parkinson's disease by intervening in α-synuclein aggregation.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. 1-Allylpiperazine in the preparation of drugs for the prevention and treatment of Parkinson's disease.

2. The application according to claim 1, characterized in that, The 1-allylpiperazine prevents and treats Parkinson's disease by inhibiting the aggregation of α-synuclein.

3. The application according to claim 2, characterized in that, The 1-allylpiperazine inhibits the aggregation of α-synuclein by binding to it, disrupting the β-sheet structure, and reducing its stability.

4. The application according to claim 1, characterized in that, The 1-allylpiperazine prevents and treats Parkinson's disease by antagonizing α-synuclein-induced cytotoxicity.

5. The application according to claim 1, characterized in that, The 1-allylpiperazine prevents and treats Parkinson's disease by interfering with the liquid-liquid phase separation of α-synuclein.

6. The application according to any one of claims 2 to 5, characterized in that, The concentration of the 1-allylpiperazine used is above 25 µM.

7. The application according to claim 1, characterized in that, The dosage form of the drug includes one or more of the following: oral preparations, injectable preparations, powders, granules, powders, and tablets.

8. A drug for the prevention and treatment of Parkinson's disease, characterized in that, Including 1-allylpiperazine.

9. The medicament according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers.

10. The medicament according to claim 8 or 9, characterized in that, The dosage form of the drug includes one or more of the following: oral preparations, injectable preparations, powders, granules, powders, and tablets.

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