Application of small molecule compound in preparation of medicine for treating and / or preventing alpha-synuclein disease

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a small molecule compound to preparation of a medicine for treating and / or preventing alpha-synuclein diseases, and the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295. The small molecule compound provided by the embodiment of the invention can accurately interfere the interaction of alpha-syn-VAPB in a targeted manner and improve neuron damage caused by abnormal aggregation of alpha-synuclein, so that alpha-synuclein diseases, especially Parkinson's disease, are safely and effectively treated and / or prevented.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to the use of small molecule compounds in the preparation of drugs for the treatment and / or prevention of α-synuclein diseases. Background Technology

[0002] Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease. Although the pathogenesis of PD is complex and influenced by multiple factors including genes, environment, and other neurodegenerative diseases, its typical pathological feature is the presence of pathological changes in α-synuclein (α-syn) in central and peripheral tissues. Specific pathological changes in α-syn include: ① abnormal folding and aggregation of α-syn protein; ② abnormal post-translational modifications of α-syn; and ③ intercellular dissemination of pathological α-syn. Numerous clinical studies have shown that as early as 20 years before a PD diagnosis, pathologically altered α-syn aggregates have been detected in nerve fibers of peripheral tissues such as the intestines and submandibular glands. Therefore, exploring the causes of α-syn pathological changes, screening for drugs to inhibit these changes, and protecting neurons from damage caused by α-syn pathological changes are of great significance for the intervention and treatment of PD.

[0003] In related technologies, traditional clinical diagnosis and treatment of Parkinson's disease focuses on the replacement and regulation of the dopaminergic system, such as dopamine replacement drugs or dopamine receptor agonists. However, these drugs can only relieve symptoms but cannot slow disease progression. Long-term use can easily cause various side effects such as nausea, vomiting, hallucinations, and impulse control disorders, and seriously affect patients' medication adherence and quality of life. Recently, interventions and drugs targeting abnormal α-synuclein aggregation have become a research focus. For example, there are currently α-synuclein aggregation inhibitors that bind to α-synuclein monomers, stabilize their native conformation, and inhibit the formation of misfolded and oligomerized α-synuclein; α-synuclein antibody drugs that specifically bind to misfolded α-synuclein, promoting its phagocytosis and clearance by macrophages or microglia; and drugs that enhance autophagy by activating the autophagy pathway to accelerate the degradation of abnormally aggregated α-synuclein. However, these still have drawbacks such as poor targeting, large molecular weight, extremely low blood-brain barrier penetration, non-specific autophagy affecting normal cellular physiological metabolism, and limitations in drug delivery methods.

[0004] Therefore, there is an urgent need to develop a small molecule compound that can precisely target the abnormal aggregation of α-synuclein, efficiently penetrate the blood-brain barrier, and has both therapeutic and safety properties for use in the preparation of drugs for the treatment and / or prevention of α-synuclein diseases, especially Parkinson's disease. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the related art.

[0006] Therefore, embodiments of the first aspect of the present invention provide the use of a small molecule compound in the preparation of a medicament for treating and / or preventing α-synuclein diseases, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295. The small molecule compound provided by the embodiments of the present invention can precisely target and interfere with α-syn-VAPB interactions, improve neuronal damage caused by abnormal aggregation of α-synuclein, thereby safely and effectively treating and / or preventing α-synuclein diseases, particularly Parkinson's disease.

[0007] In some embodiments, the α-synuclein disease is at least one of Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0008] In some embodiments, the α-synuclein disease is Parkinson's disease.

[0009] In some embodiments, the small molecule compound targets and inhibits the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B.

[0010] In some embodiments, the small molecule compound improves neuronal damage.

[0011] In some embodiments, the application concentration of the small molecule compound ranges from 1 nM to 100 μM.

[0012] An embodiment of the second aspect of the present invention provides the use of a small molecule compound in the preparation of a medicament for improving and / or preventing neuronal damage, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295.

[0013] In some embodiments, the neuronal damage is neuronal damage caused by an excess of α-synuclein.

[0014] An embodiment of the third aspect of the present invention provides a pharmaceutical composition for treating and / or preventing α-synuclein diseases, comprising at least one small molecule compound selected from AO-365 / 43264238 and AG-670 / 40728295 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient, carrier or diluent.

[0015] In some embodiments, the small molecule compound targets and inhibits the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B, thereby improving neuronal damage.

[0016] An embodiment of the fourth aspect of the present invention provides the use of a small molecule compound in targeting and inhibiting the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295.

[0017] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0018] This invention provides for the first time small molecule compounds AO-365 / 43264238 and / or AG-670 / 40728295 based on the mechanism of targeting and interfering with the interaction between α-synuclein and VAPB, for use in the preparation of drugs for the treatment and / or prevention of α-synuclein diseases (especially Parkinson's disease). The small molecule compound has the following advantages: (1) It is convenient to take and can be administered in various ways, such as direct oral administration without invasive administration; (2) It has a small molecular weight and simple structure, strong tissue penetration and lipid solubility, and can easily penetrate the blood-brain barrier to achieve targeted drug delivery; (3) It has a mature synthesis process, low cost, stable chemical properties, and does not require cold chain transportation and low temperature storage, so it is easy to promote and apply in clinical practice; (4) Small molecule drugs are usually non-immunogenic, thus avoiding the risk of reduced efficacy or allergies caused by immune reactions, and they are metabolized quickly, so even if adverse reactions occur, they will not accumulate in the body for a long time; (5) It effectively targets and inhibits the interaction between α-synuclein and vesicle-associated membrane protein-associated protein B, and improves neuronal damage, thereby treating and / or preventing α-synuclein diseases. Attached Figure Description

[0019] Figure 1 A schematic diagram illustrating key interaction features when searching for small molecule compounds in embodiments of the present invention based on virtual screening is shown.

[0020] Figure 2 The diagram illustrates the principle and results of using affinity analysis experiments based on biomembrane interference technology to screen small molecule compounds according to embodiments of the present invention.

[0021] Figure 3 The figure shows the effect of small molecule compounds from embodiments of the present invention on the α-syn aggregation process.

[0022] Figure 4 The figure shows the effect of small molecule compound treatment on neuronal damage caused by α-syn overexpression according to an embodiment of the present invention.

[0023] Figure 5 The figure shows the effect of small molecule compound treatment on α-syn-VAPB interaction in cells according to an embodiment of the present invention. Detailed Implementation

[0024] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0025] This invention is based on the inventor's discoveries and understanding of the following facts and problems: Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease. Although the pathogenesis of PD is complex and influenced by multiple factors including genes, environment, and other neurodegenerative diseases, its typical pathological feature is the presence of pathological changes in α-synuclein (α-syn) in central and peripheral tissues. Specific pathological changes in α-syn include: ① abnormal folding and aggregation of α-syn protein; ② abnormal post-translational modifications of α-syn; and ③ intercellular dissemination of pathological α-syn. Numerous clinical studies have shown that as early as 20 years before a PD diagnosis, pathologically altered α-syn aggregates have been detected in nerve fibers of peripheral tissues such as the intestines and submandibular glands. Therefore, exploring the causes of α-syn pathological changes and screening for processes that inhibit these changes is of great significance for the intervention and treatment of PD.

[0026] Furthermore, diseases directly related to abnormal α-synuclein aggregation are collectively referred to as α-synucleinopathies. These diseases share the common pathological feature of misfolded α-synuclein oligomers, fibrils, and characteristic deposits such as Lewy bodies / Lewy neurites, and glial cytoplasmic inclusions within the central or peripheral nervous system, leading to neuronal or glial cell damage and nervous system dysfunction. Common α-synucleinopathies include Lewy body dementia, multiple system atrophy, pure autonomic failure, and Parkinson's disease dementia. Abnormal α-synuclein aggregation is one of the driving factors in the development and progression of these diseases. Therefore, research into the mechanisms of abnormal α-synuclein aggregation and the development of targeted interventions are not only of great value in alleviating symptoms and slowing the progression of Parkinson's disease, but also provide a reference for elucidating the pathogenic mechanisms of other α-synucleinopathies.

[0027] Regarding the pathological changes of α-syn, previous studies have identified proteins interacting with α-syn in the brain tissue of various PD transgenic mouse models from the perspective of identifying interacting proteins. These classic interacting proteins include: Vesicle-Associated Membrane Protein 2 (VAMP2), Syntaxin, Synaptosomal-Associated Protein 25 (SNAP-25), Synphilin-1, Cysteine ​​String Protein α (CSPα), Tau protein, Heat Shock Protein 70 (Hsp70), Heat Shock Protein 90 (Hsp90), Neurogranin, Amyloid Precursor Protein (APP), and β-Amyloid (Aβ). Furthermore, previous studies have found an interaction between α-syn and vesicle-associated membrane protein-associated protein (VAPB) in the mitochondria-associated endoplasmic reticulum (MAM) region. Building upon this, our research team further discovered that phosphorylation at serine 129 of α-syn increases the interaction between α-syn and the VAPB and protein tyrosine phosphatase interacting protein 51 (PTPIP51) scaffolding complex in the MAM region. VAPB is a highly conserved membrane protein primarily distributed in the endoplasmic reticulum. Research on VAPB has mainly focused on amyotrophic lateral sclerosis (ALS), but in 2015, scientists identified a deletion mutation of a valine residue at position 25 (p.ΔV25) in VAPB from a sporadic PD patient. Subsequently, using immunohistochemistry and immunoelectron microscopy, researchers discovered that α-syn and VAPB were co-localized in pathological aggregate structures within the substantia nigra neurons of PD patients. Moreover, compared to the control group, the VAPB protein content in neurons was significantly reduced.The above results suggest that VAPB is involved in the occurrence and development of PD, but the specific mechanisms still need to be explored in depth.

[0028] However, there is currently a lack of research on whether interfering with α-syn-VAPB interactions under pathological conditions can block abnormal interactions between α-syn and membranous organelles and thus exert a neuroprotective effect. Furthermore, no small molecule compounds that interfere with α-syn-VAPB interactions have been developed domestically or internationally.

[0029] ADMET is a key technology in drug development used to evaluate the absorption, distribution, metabolism, excretion, and toxicity of drugs in vivo, and it is of great significance to drug safety and efficacy. In the early stages of drug development, targeted selection and optimization of lead compounds based on their ADMET properties can significantly improve the success rate of drug development and reduce the waste of funds caused by ADMET property issues in later stages of drug development.

[0030] Bio-Layer Interferometry (BLI) is a label-free optical biosensing technology widely used for the real-time monitoring and analysis of biomolecular interactions. In principle, when two or more light waves meet in space, they superimpose; this phenomenon is called light interference. The BLI molecular interaction analyzer utilizes this light interference phenomenon. The bottom of the biosensor on the analyzer is covered by a biofilm layer, which can bind or immobilize biomolecules. When a beam of visible light is incident perpendicularly on the biofilm layer, the light is reflected at the two interfaces of the biofilm layer, forming an interference wave of a specific wavelength. When immobilized molecules interact with molecules in the solution, the thickness of the biofilm layer increases, and the interference spectrum shifts towards increasing wavelength. This phase shift of the light wave is detected in real time by a detector. By analyzing the phase shift of the light wave, the changes in the number of molecules on the sensor surface and related concentration and kinetic data can be quantitatively obtained.

[0031] This invention employs molecular dynamics simulations and virtual screening to identify small molecule compounds that interfere with the α-syn-VAPB interaction. Subsequently, biomembrane interference (BLI) technology narrows the screening to the top three small molecule compounds with the highest inhibitory effects. ThT and Western blotting experiments are then used to detect the effects of these small molecule compounds on the in vitro aggregation process of shaken α-syn protein, identifying compounds AO-365 / 43264238 and AG-670 / 40728295 as inhibitors of this aggregation process. Furthermore, the effects of AO-365 / 43264238 and AG-670 / 40728295 on cell viability are examined in an α-syn-overexpressing neuronal cell model, further validating the effectiveness of the screened small molecule compounds in interfering with the α-syn-VAPB interaction. Therefore, this invention provides for the first time two small molecule compounds with potential application value for use in the preparation of drugs for treating and / or preventing α-synuclein diseases, especially Parkinson's disease. These compounds can precisely target and interfere with α-syn-VAPB interactions, improve neuronal damage caused by abnormal aggregation of α-synuclein, and thus safely and effectively treat and / or prevent α-synuclein diseases.

[0032] An embodiment of the first aspect of the present invention provides the use of a small molecule compound in the preparation of a medicament for treating and / or preventing α-synuclein diseases, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295. The small molecule compound provided by the embodiments of the present invention can precisely target and interfere with α-syn-VAPB interactions, improve neuronal damage caused by abnormal aggregation of α-synuclein, thereby safely and effectively treating and / or preventing α-synuclein diseases, particularly Parkinson's disease.

[0033] In some specific embodiments, AO-365 / 43264238 is shown in Equation 1, and AG-670 / 40728295 is shown in Equation 2: Formula 1, Equation 2.

[0034] In some embodiments, the α-synuclein disease is at least one of Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0035] In some embodiments, the α-synuclein disease is Parkinson's disease.

[0036] In some embodiments, the small molecule compound targets and inhibits the interaction between α-synuclein (UniProt: P37840) and vesicle-associated membrane protein-associated protein B (UniProt: O95292).

[0037] In some embodiments, the small molecule compound improves neuronal damage.

[0038] In some embodiments, the application concentration of the small molecule compound ranges from 1 nM to 100 μM.

[0039] An embodiment of the second aspect of the present invention provides the use of a small molecule compound in the preparation of a medicament for improving and / or preventing neuronal damage, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295.

[0040] In some embodiments, the neuronal damage is neuronal damage caused by an excess of α-synuclein.

[0041] An embodiment of the third aspect of the present invention provides a pharmaceutical composition for treating and / or preventing α-synuclein diseases, comprising at least one small molecule compound selected from AO-365 / 43264238 and AG-670 / 40728295 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient, carrier or diluent.

[0042] In some embodiments, the small molecule compound targets and inhibits the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B, thereby improving neuronal damage.

[0043] An embodiment of the fourth aspect of the present invention provides the use of a small molecule compound in targeting and inhibiting the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295.

[0044] The small molecule compounds provided in this invention have the following advantages when used for the prevention and / or treatment of Parkinson's disease: (1) They are convenient to take and can be administered in various ways, such as direct oral administration without invasive administration; (2) They have small molecular weight, simple structure, strong tissue penetration and lipid solubility, and can easily penetrate the blood-brain barrier to achieve targeted drug delivery; (3) They have mature synthesis processes, low cost, stable chemical properties, and do not require cold chain transportation and low temperature storage, so they are easy to promote and apply in clinical practice; (4) Small molecule drugs are usually non-immunogenic, thus avoiding the risk of reduced efficacy or allergies caused by immune reactions, and they are metabolized quickly, so even if adverse reactions occur, they will not accumulate in the body for a long time; (5) They effectively target and inhibit the interaction between α-synuclein and vesicle-associated membrane protein-associated protein B and improve neuronal damage, thereby treating and / or preventing α-synuclein diseases.

[0045] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0046] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] Unless otherwise specified, the quantitative analysis experiments in the following examples are all performed in triplicate, and the results are averaged.

[0048] Example 1: Screening of candidate small molecule compounds targeting and inhibiting α-syn-VAPB interactions In this embodiment, AO-365 / 43264238, AF-399 / 37305022 and AG-670 / 40728295 were selected as candidate small molecule compounds through virtual screening (virtual docking and ADMET screening) and BLI technology for subsequent verification.

[0049] 1.1 Using virtual screening methods to find candidate small molecule compounds In this embodiment, a virtual simulation was used to screen out the top 10 small molecule compounds that have the potential to inhibit α-syn-VAPB interactions.

[0050] (1) Determine the interaction sequence of α-syn-VAPB.

[0051] First, the human α-syn-VAPB-PTPIP51 complex in equilibrium was obtained using molecular dynamics simulations, and the interaction sequence of α-syn-VAPB in the complex was identified.

[0052] (2) Docking simulation.

[0053] Using the VAPB-MSP dimer structure (PBD name: 3IKK) in the complex (MSP being the N-terminal major spermin domain where α-syn interacts primarily with the VAPB protein) as the receptor, the region of VAPB that binds to h-α-syn was selected as the docking pocket. Approximately 210,000 molecules from the Specs molecular library (https: / / www.specs.net / ) were docked to the VAPB docking pocket. The docked molecules are characterized by ligand centers, and the docking simulation was based on the shape and geometric distance matching between the receptor pocket center and the ligand center.

[0054] (3) Evaluate the docking simulation score to conduct the first round of screening of candidate small molecule compounds.

[0055] All compounds were treated to remove salts and free ions, and then their 3D structures were generated using RDKit and used as ligands for docking simulations. All docking simulations were performed using the DOCK6 program (University of California San Francisco, version 6.10; Allen WJ, Balius TE, Mukherjee S, et al. DOCK 6: Impact of new features and current docking performance. J ComputChem 2015; 36: 1132-1156). The docked molecules were characterized by ligand centers, and the docking simulations were based on shape and geometric distance matching between the acceptor pocket center and the ligand center. Hawkins-Cramer-Truhlar GB / SA and PB / SA scores, as well as the AMBER molecular mechanics score function with implicit solvents, were implemented using OpenEye's Zap library. The conformations of the complexes were evaluated through multi-dimensional scoring. High-throughput rigid docking simulations were performed to rank all compounds, and semi-flexible docking was performed on the top 10,000 compounds for rigid docking. The 10,000 compounds were ranked based on their semi-flexible docking scores, and then cluster analysis based on molecular fingerprinting was performed. The compound with the highest docking score in each cluster was selected as the representative molecule, and the top 1,000 compounds by docking score after clustering were obtained.

[0056] (4) A second round of screening of candidate small molecule compounds was conducted using ADMET prediction.

[0057] ADMET prediction data of candidate small molecule compounds were obtained through ADMET prediction on the Discovery Studio platform, and a second round of screening was conducted based on the following criteria: ① Solubility level: 2 or 3 (soluble); ② Blood-brain barrier penetration ability: 0, 1, 2 or 3 (penetrating); ③ No inhibition of CYP2D6 enzyme activity; ④ No hepatotoxicity; ⑤ Absorption capacity: 0 (moderate absorption); ⑥ No binding to plasma proteins.

[0058] Figure 1 This diagram illustrates key interaction features when searching for small molecule compounds according to embodiments of the present invention using virtual screening. Figure 1 Part A is a diagram of the optimal interaction patterns of the proteins in the α-syn-VAPB-PTPIP51 complex (where the A chain of VAPB (VAPB_A) is shown in yellow, the B chain of VAPB (VAPB_B) is shown in cyan, PTPIP51 is shown in green, and h-α-syn is shown in magenta; key residues in VAPB_A are shown in yellow bars, key residues in VAPB_B are shown in cyan bars, key residues in PTPIP51 are shown in green bars, and key residues in h-α-syn are shown in magenta bars). Figure 1 Part B is the interaction sequence between α-syn and VAPB in the complex; Figure 1 Part C is a schematic diagram of the binding sites of VAPB_A and h-α-syn. The area marked by the black box in the figure is the docking pocket region where the two bind.

[0059] The top 10 candidate small molecule compounds obtained through the virtual screening method above in terms of docking scores are: AG-670 / 40727464, AE-641 / 15339362, AO-365 / 43264238, AF-399 / 37305022, AG-670 / 40887795, AJ-292 / 14921086, AG-670 / 40728295, AE-641 / 00585062, AF-399 / 14425111, and AN-465 / 42246715. These compounds have the potential to inhibit α-syn-VAPB interactions.

[0060] 1.2 Using BLI technology to find candidate small molecule compounds In this embodiment, the 10 candidate small molecule compounds were tested using the BLI experiment, and three small molecule compounds with potential inhibitory effects on α-syn-VAPB interaction were screened.

[0061] (1) Biotinylation labeling of human α-syn protein (h-α-syn) The full-length h-α-syn protein was purified using an E. coli prokaryotic protein expression vector. First, the recombinant plasmid pGEX-6P-1-GST-SNCA (Heyuan Biotechnology) was transformed into E. coli BL21 competent cells (Tiangen Biotech, CB105-01). After amplification of the engineered bacteria, the expression of the fusion protein was induced by 0.1 mM Isopropyl-β-D-1-Thiogalactopyranoside (Beyotime, ST098). After collecting the bacterial lysate, the GST-h-α-syn protein was enriched using a GST-tagged protein purification kit (Beyotime, P2262). Subsequently, the h-α-syn protein (1 mg / mL, 1-140AA) was obtained by cleavage with PreScission Protease (Beyotime, P2302). Subsequently, it was mixed and incubated with EZ-Link™ NHS-LC-LC-biotin (10 mg / mL, Themro, 21343), and unbound biotin was removed using a desalting column (Themro, 89882).

[0062] (2) Biotinylated h-α-syn (Bio-h-α-syn, 10 μg / mL) protein was immobilized on the SA biosensor of the Gator label-free biomolecular analysis system (Beijing Top Biotech Co., Ltd.)

[0063] (3) Human VAPB-MSP-His protein (VAPB / MSP, 1-132AA, Sinocare, 10754-H08E) was diluted sequentially with PBS to 1600, 800, 400, 200, 100, 50 and 25 nM solutions, and then affinity analysis was performed using the Gator label-free biomolecular analysis system.

[0064] (4) Referring to steps (1)-(2), biotinylated h-α-syn (Bio-h-α-syn, 10 μg / mL) is immobilized on the SA biosensor of the Gator analyzer.

[0065] (5) Referring to step (3), VAPB was diluted to 800 nM using PBS, and then 10 small molecule compounds obtained in Example 1.1 were added according to the group to make a final concentration of 1 mM, so as to detect the inhibitory effect of the compounds on the h-α-syn-VAPB interaction by affinity analysis experiment.

[0066] The results are as follows Figure 2 As shown. Figure 2The diagram illustrates the principle and results of affinity analysis experiments based on biomembrane interferometry to screen small molecule compounds according to embodiments of the present invention. Figure 2 Part A is a schematic diagram of the principle of biomembrane interference (BLI) technology; Figure 2 Part B shows the results of the affinity analysis between VAPB protein and h-α-syn protein. Figure 2 Part C is the result of the affinity analysis experiment between VAPB protein and h-α-syn protein after treatment with the small molecule compound AF-399 / 37305022; Figure 2 Part D is the result of the affinity analysis experiment between VAPB protein and h-α-syn protein after treatment with the small molecule compound AO-365 / 43264238; Figure 2 The E part represents the results of the affinity analysis between VAPB protein and h-α-syn protein after treatment with the small molecule compound AG-670 / 40728295.

[0067] from Figure 2 As shown in Part A, when different concentrations of VAPB bind to Bio-h-α-syn immobilized on the biosensor surface, the thickness of the optical layer increases, resulting in a longer path length for the second reflected light. This causes a change in the interference spectrum curve, shifting it to the right. When VAPB and Bio-h-α-syn dissociate, the molecules dissociate from the biosensor surface into the solution, causing the interference spectrum curve to shift to the left. By plotting the sensor map with the offset distance of the interference spectrum curve versus the reaction time, the binding constant (Kon), dissociation constant (Koff), and affinity constant (KD) of VAPB and Bio-h-α-syn can be fitted based on the binding model.

[0068] from Figure 2 As shown in Part B, different concentrations of VAPB solution exhibit significant binding signals with Bio-h-α-syn solidified on the probe, demonstrating a strong concentration dependence. The signal converges at high concentrations, indicating that the concentration is close to or has reached saturation, suggesting specific binding. The binding constant (K0) is also relevant. on = 5.85 x 10 3 M -1 S -1 ; dissociation constant (K off = 7.92 x 10 -4 S -1 Affinity constant (KD) = 1.35 x 10⁻⁶ -7 M; Full R 2 = 0.987.

[0069] This embodiment utilizes the BLI method described above to screen for the top three small molecule compounds that inhibit α-syn-VAPB interactions, and then... Figure 2 The CE section specifically illustrates the affinity analysis results of VAPB protein and h-α-syn protein after treatment with small molecule compounds. The upper part shows the chemical structure diagram of the small molecule compound, and the lower part shows the signal fitting sensor diagram (black curve: control group; orange curve: group treated with small molecule compound; immobilized protein: biotinylated h-α-syn (Bio-h-α-syn, 10 μg / mL); mobile phase: VAPB-MSP-His protein (800 nM); final small molecule concentration: 1 mM). It can be seen that all three small molecule compounds effectively inhibited the α-syn-VAPB interaction in vitro.

[0070] Example 2: Effect of small molecule compound treatment on α-syn aggregation process In this embodiment, the effects of the small molecule compounds obtained in Example 1 on the α-syn aggregation process were detected by Western blotting and ThT staining, and it was determined that AO-365 / 43264238 and AG-670 / 407282955 reduced the content of β-sheet structures in α-syn aggregates.

[0071] (1) Prepare 150 μL reaction systems (control group; AF-399 / 37305022 treatment group; AO-365 / 43264238 treatment group; AG-670 / 407282955 treatment group), including 20 μL of small molecule compound (mother liquor concentration: 20mM), 130 μL of h-α-syn monomer (1 mg / mL), and 1.5 μL of preservative (Beyotime, ProClean 300, ST853).

[0072] (2) The reaction system was then placed vertically on a horizontal shaker at 37°C and shaken continuously at 1000 rpm for 7 days to induce the formation of α-syn monomeric proteins into α-syn prefibrils that induce pathological spread through in vitro physical shaking. These prefibrils contain β-sheet structures similar to those in the pathological LBs of PD patients' brain tissue.

[0073] (3) After denaturation of the shaken product, the effects of three small molecule compounds on the formation of α-syn aggregates were detected by Western blotting. The specific procedure was as follows: The shaken protein product was mixed with loading buffer (250 mM Tris-HCl, pH 6.8, 30% glycerol, 0.02% Bromophenol Blue) and denatured at 95ºC for 10 min. The denatured protein product was loaded at a dose of 200 ng per lane, and the protein samples were separated by 12% SDS-PAGE electrophoresis. The proteins on the gel were transferred at 2 mA / cm using a semi-dry transfer method. 2 The transfer rate was rapidly increased to a 0.22 μm polyvinylidene fluoride (PVDF, Millipore, ISEQ00010) membrane, with a transfer time of 90 min. The membrane was then blocked with 5% skim milk at room temperature for 1 h, followed by overnight incubation with h-α-syn primary antibody dilution buffer (Abcam, ab138501, 1 / 2,000). After primary antibody recovery, the membrane was washed and then incubated with Rabbit 680 secondary antibody dilution buffer (LI-CORBiosciences, 926-68071, 1 / 10,000) at room temperature for 1 h. The membrane was then washed again, and the images were developed and photographed using an Odyssey imaging system (LI-COR Biosciences).

[0074] (4) After denaturing the shake product, the effects of the three small molecule compounds on the content of β-sheet structure in the aggregate were detected by thiosulfate S (ThT, Sigma, T3516) staining method (refer to the instruction manual for specific detection methods).

[0075] Test results as follows Figure 3 As shown. Figure 3 The figure shows the effect of small molecule compounds from embodiments of the present invention on the α-syn aggregation process.

[0076] Depend on Figure 3As can be seen in Part A, in the control group lanes, in addition to the band appearing at the h-α-syn monomer (17 kD) position, tailing bands were formed in the sample wells and in lanes above 17 kD, proving that in vitro physical shaking formed α-syn aggregates. In the AF-399 / 37305022 treatment group, trailing bands with higher gray values ​​were formed in the sample wells and lanes above 17 kD, indicating that AF-399 / 37305022 promoted the aggregation of α-syn during shaking. In the AO-365 / 43264238 treatment group, trailing bands with lower gray values ​​were formed in the sample wells and lanes above 17 kD, indicating that AO-365 / 43264238 inhibited the aggregation of α-syn during shaking. In the AG-670 / 40728295 treatment group, trailing bands with lower gray values ​​were formed in the sample wells and lanes above 17 kD, indicating that AG-670 / 40728295 inhibited the aggregation of α-syn during shaking.

[0077] Similar to the results of Western blotting experiments, the ThT staining results of the shaken products showed that... Figure 3 As shown in Part B, compared with the control group, AF-399 / 37305022 promoted the content of β-sheet structures in α-syn aggregates, while AO-365 / 43264238 and AG-670 / 40728295 reduced the content of β-sheet structures in α-syn aggregates. Figure 3 Sections C and D illustrate the interaction modes of AO-365 / 43264238 and AG-670 / 40728295 with VAPB.

[0078] Example 3: Effects of small molecule compound treatment on neuronal damage induced by α-syn overexpression In this embodiment, cells were first treated with AO-365 / 43264238 and AG-670 / 40728295 to determine that the compounds themselves did not have cytotoxicity. Then, the effects of the compounds on neuronal damage caused by α-syn overexpression were investigated in an α-syn overexpression cell model. It was determined that AO-365 / 43264238 and AG-670 / 40728295 alleviated the cytotoxicity caused by α-syn overexpression to a certain extent, thereby exerting a neuroprotective effect.

[0079] 3.1 Investigation of the cytotoxicity of the compound (1) The small molecule compounds AO-365 / 43264238 and AG-670 / 40728295, which were serially diluted to final concentrations of 1 nM, 10 nM, 100 nM, 1000 nM, 10 μM and 100 μM, were added to the SHSY5Y cell line (Chinese Academy of Sciences Cell Bank, BFN60700126). After treatment for 24 h, cell viability was detected using the CCK8 kit (Beyotime, C0042).

[0080] (2) The small molecule compounds AO-365 / 43264238 and AG-670 / 40728295, which were serially diluted to final concentrations of 1 nM, 10 nM, 100 nM, 1000 nM, 10 μM and 100 μM, were added to primary mouse cortical neurons cultured in vitro. After treatment for 72 h, cell viability was detected using a CCK8 assay kit.

[0081] 3.2 Effects of small molecule compound treatment on neuronal damage induced by α-syn overexpression (1) The recombinant pCMV-myc-h-α-syn plasmid (Heyuan Biotechnology) was transiently transfected into the SHSY5Y cell line using PEI transfection reagent Transporter 5 (Polysciences, 26008-1A). The cells were then treated for 24 h. Mouse primary cortical neuronal cells were infected with Ubi-MCS-3FLAG-SNCA-SV40-puromycin (Shanghai Jikai Gene Co., Ltd.) for 4 days to prepare an α-syn overexpression cell model.

[0082] (2) AO-365 / 43264238 and AG-670 / 40728295 at 10 and 50 μM were used to treat the α-syn overexpression cell model based on SHSY5Y cell line for 24 hours; and the α-syn overexpression cell model based on mouse cortical primary neurons for 72 hours. Cell viability was then detected using the CCK8 kit.

[0083] The results are as follows Figure 4 As shown. Figure 4 The figure shows the effect of small molecule compound treatment on neuronal damage caused by α-syn overexpression according to embodiments of the present invention (statistical methods used were one-way ANOVA and Tukey's multiple comparisons, where AB. n = 3, C. n = 6, DE. n = 4, F. n = 8). P<0.01, P<0.001, P<0.0001 vs. Con. #P<0.05, ##P<0.01 vs. SNCA.).

[0084] Depend on Figure 4 As shown in sections A, B, D, and E, AO-365 / 43264238 and AG-670 / 40728295 at final concentrations of 1 nM, 10 nM, 100 nM, 1000 nM, 10 μM, and 100 μM did not exhibit cytotoxicity.

[0085] Depend on Figure 4 As shown in sections C and F, α-syn overexpression significantly increased cytotoxicity, while treatment with 10 / 50 μM AG-670 / 40728295 alleviated the cytotoxicity caused by α-syn overexpression to some extent. Therefore, AO-365 / 43264238 and AG-670 / 40728295 can exert neuroprotective effects by interfering with the α-syn-VAPB interaction, thereby improving neuronal damage caused by α-syn overexpression.

[0086] Example 4: Effects of small molecule compound treatment on α-syn-VAPB interactions in cells (1) Recombinant pCMV-myc-h-α-syn plasmid (Heyuan Biotechnology) was transiently transfected into SHSY5Y cell line using PEI transfection reagent Transporter 5 (Polysciences, 26008-1A) for 24 h. Then, AO-365 / 43264238 and AG-670 / 40728295 (final concentration: 50 μM) were added for 24 h. Cells were then collected and cellular protein components were extracted using IP buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-Na2, 1% NP-40, 1X protease inhibitor and phosphatase inhibitor mixture). After sonication on ice, the cells were centrifuged at 12,000 g for 30 min at 4°C. The supernatant was collected for CO-IP and Western Blot experiments. Protein concentration in cell extracts was determined using a BCA kit (Thermo, 23225). Subsequently, 1,000 μg of cell protein solution, h-α-syn antibody (Abcam, ab138501, 1 / 200) used in the CO-IP experiment, and control antibody IgG (CST, 2729S) were added to 400 μL of the IP system (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-Na2, 1% NP-40, 1X protein inhibitor and phosphatase inhibitor). The CO-IP system was continuously shaken and mixed in a vertical shaker at 4°C for 16 h. Then, 30 μL of Protein A / G Magnetic Beads (MCE, HY-K0202) were added, and the mixture was continuously shaken and mixed in a vertical shaker at 4°C for 6 h. The bead-antigen-antibody complexes were then separated using a magnetic rack. 40 μL of the output component was mixed with 10 μL of 5X loading buffer, and the bead-antigen-antibody complex was mixed with 30 μL of 2X loading buffer. The mixture was then heated at 95ºC for 10 min to denature the mixture. The prepared CO-IP protein sample was then detected by Western blotting.

[0087] (2) Perform Western Blot analysis on CO-IP samples.

[0088] The primary antibody information used in the Western blot experiment is as follows: h-α-syn (Abcam, ab138501, 1 / 2,000), p-α-syn (CST, #23706, 1 / 2,000), VAPB (Proteintech, 14477-1-AP, 1 / 1,000), β-actin (ABclonal, AC026, 1 / 4,000), and GAPDH (ABclonal, A19056, 1 / 5,000).

[0089] The results are as follows Figure 5 As shown. Figure 5 The figure shows the effect of small molecule compound treatment on α-syn-VAPB interaction in cells according to an embodiment of the present invention.

[0090] Depend on Figure 5 As can be seen from Part A, the small molecule compounds AO-365 / 43264238 and AG-670 / 40728295 do not affect the content of VAPB and h-α-syn protein.

[0091] Depend on Figure 5 As shown in sections B and C, the CO-IP results revealed that, compared to the control group (α-syn + Con), the AG-670 / 40728295 treatment group (α-syn + AG-670) exhibited a significant decrease in the content of VAPB proteins interacting with α-syn, while the corresponding output component showed an increase in VAPB protein content. This indicates that AG-670 / 40728295 significantly inhibited the interaction between α-syn and VAPB in the SHSY5Y cell line. In contrast, the effect of AO-365 / 43264238 was not significant, but it still showed a trend of inhibiting the interaction between α-syn and VAPB in the SHSY5Y cell line.

[0092] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0093] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0094] References 1. Allen WJ, Balius TE, Mukherjee S, et al. DOCK 6: Impact of new features and current docking performance. J Comput Chem 2015; 36: 1132-1156.

Claims

1. The use of a small molecule compound in the targeted inhibition of the interaction between α-synuclein protein and vesicle-associated membrane protein-associated protein B, wherein the small molecule compound is selected from at least one of AO-365 / 43264238 and AG-670 / 40728295. The application concentration of the small molecule compound is 50 μM.

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