Application of 9-Methylfacaplysin in preparation of medicine for treating or preventing nervous system diseases
By using 9-Methylfascaplysin as a ROCK2 inhibitor, the problems of strong side effects and weak specificity of existing drugs in promoting neurite growth and regeneration have been solved, achieving effective treatment for neurodegenerative diseases and nervous system diseases.
Patent Information
- Application Number
- CN202511665768.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-02-17
AI Technical Summary
Existing ROCK2 inhibitors have problems such as strong side effects and low specificity in promoting neurite growth and regeneration. There is an urgent need to develop more effective inhibitors to treat neurodegenerative diseases and nervous system diseases.
9-Methylfascaplysin was used as a ROCK2 inhibitor. By interacting with specific binding sites of the ROCK2 protein, it inhibited the activity of ROCK2, thereby promoting neurite growth and regeneration.
9-Methylfascaplysin significantly promotes neurite growth and regeneration, with effects similar to existing drugs while reducing side effects. It is suitable for the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and neurological diseases such as ischemic stroke.
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Figure CN121534054A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to the application of 9-Methylfascaplysin in the preparation of drugs for the treatment or prevention of nervous system diseases. Background Technology
[0002] Damage to neurite growth or regeneration is an early pathological feature of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), as well as various neurological disorders such as ischemic stroke, hemorrhagic stroke, and traumatic brain injury. In these diseases, damage to neurite growth or regeneration usually precedes neuronal loss and behavioral disorders. Under these pathological conditions, the growth and regeneration potential of damaged neurites are inhibited by the microenvironment, including neuroinflammation and oxidative stress. Therefore, it is generally believed that promoting neurite growth and regeneration through therapeutic means is a feasible strategy for treating these neurodegenerative and neurological disorders.
[0003] Rho-associated coiled-coil protein kinase 2 (ROCK2) is a key kinase in the Rho / ROCK signaling pathway, participating in the regulation of various cellular functions, including cell morphology, motility, survival, apoptosis, cytoskeleton remodeling, and neurite growth. ROCK2 phosphorylates downstream target proteins such as myosin light chain (MLC), MYPT, CRMP2, and LIM kinase (LIMK), leading to excessive stabilization of the actin cytoskeleton, thereby inhibiting neuronal growth cone guidance, axonal elongation, and neurite growth and regeneration. Therefore, ROCK2 inhibitors can be used as neurite growth and regeneration promoters to treat the aforementioned neurodegenerative and nervous system diseases. However, existing ROCK2 inhibitors suffer from strong side effects and low specificity, necessitating the development of more effective ROCK2 inhibitors to promote neurite growth and regeneration.
[0004] 9-Methylfascaplysin is a derivative of the compound fascaplysin isolated from the symbiotic bacterium Fascaplysinopsis sp. of Fiji. This application further investigates its application. Summary of the Invention
[0005] In view of the current state of the prior art, this invention provides the application of 9-Methylfascaplysin in the preparation of drugs for the treatment or prevention of neurological diseases, including neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as ischemic stroke, hemorrhagic stroke, and traumatic brain injury.
[0006] The present invention is solved by the following technical solution.
[0007] The use of 9-Methylfascaplysin in the preparation of medicaments for the treatment or prevention of neurological diseases, wherein the medicaments include one of the following: tablets, capsules, pills, injections, oral preparations, and suspensions containing 9-Methylfascaplysin.
[0008] In a preferred embodiment, the neurological disease includes at least one of the following: Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, ischemic stroke, hemorrhagic stroke, and traumatic brain injury.
[0009] The technical solution in this invention has the following beneficial effects: 9-Methylfascaplysin can promote the growth and regeneration of neurites and can be used to treat neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as various neurological diseases such as ischemic stroke, hemorrhagic stroke, and traumatic brain injury. Attached Figure Description
[0010] Figure 1 The morphological changes of PC12 cells after the addition of 9-MF and retinoic acid included: Figure 1 A represents the average length and number of neural spurs detected by FDA staining. Figure 1 B shows that quantitative analysis results indicate that 9-MF significantly induced neurite growth and regeneration in PC12 cells (compared to the control group).
[0011] Figure 2 The addition of 9-MF and retinoic acid to PC12 cells altered the growth-related protein GAP-43, among other things: Figure 2 A represents the GAP-43 positive area detected by immunocytochemical staining; Figure 2 B indicates that quantitative analysis showed that 9-MF significantly upregulated the area of GAP-43 positive regions in PC12 cells; Figure 2 C represents the detection of GAP-43 and GAPDH protein expression levels by Western blotting. Figure 2 D represents the quantitative analysis of Western blotting results.
[0012] Figure 3 Mitochondrial changes occurred in PC12 cells after the addition of 9-MF and retinoic acid, including: Figure 3 A represents the mitochondrial area detected by Mito-tracker staining. Figure 3 B indicates that quantitative analysis showed that 9-MF significantly increased the mitochondrial area of PC12 cells.
[0013] Figure 4 The volcano plot shows the differential RNA-seq expression results of PC12 cells in the 9-MF treatment group and the control group.
[0014] Figure 5 GO cell component analysis showed that 9-MF-related differentially expressed genes were significantly enriched in mitochondrial and synaptic structures.
[0015] Figure 6 KEGG pathway analysis showed that 9-MF-related DEGs were enriched in various neurodegenerative and nervous system disease pathways.
[0016] Figure 7 Protein interaction network analysis revealed that 9-MF-related DEGs form a regulatory network with ROCK2 as the core.
[0017] Figure 8 The Venn diagram shows the intersection of 9-MF-related DEGs with genes related to neurite growth and regeneration.
[0018] Figure 9 for Figure 8 GO molecular function analysis of 9-MF-related DEGs revealed significant enrichment in kinase activity and microtubule binding function.
[0019] Figure 10 This is the result of CMap analysis.
[0020] Figure 11 The dot plots show the binding affinity of different ROCK2 inhibitors to ROCK2, with the size and color intensity of the dots corresponding to the strength of the binding affinity.
[0021] Figure 12 To elucidate the optimal binding conformation of 9-MF and ROCK2 for molecular docking analysis.
[0022] Figure 13 The addition of 9-MF and fasudil to PC12 cells resulted in changes in p-ROCK2 levels, including: Figure 13 A: PC12 cells were treated with 9-MF and fasudil for 0.5 hours, respectively, and the p-ROCK2 positive area was detected by immunocytochemical staining. Figure 13 B represents the results of quantitative analysis: 9-MF significantly reduced the area of p-ROCK2 positive regions in PC12 cells (p < 0.01 compared with the control group). Figure 13 C represents the detection of p-ROCK2 and ROCK2 protein expression levels by Western blotting; Figure 13 D represents the quantitative analysis of Western blotting results.
[0023] Figure 14 The morphological changes of PC12 cells after treatment with 9-MF and narcissin for 48 hours were shown, including: Figure 14 A is FDA staining. Figure 14 B represents the results of quantitative analysis: 9-MF significantly promoted neurite growth and regeneration (p < 0.01 vs control group), while narcissin could reverse this effect (#p < 0.05 vs 9-MF group). Detailed Implementation
[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0025] 9-Methylfascaplysin is a derivative of the compound fascaplysin isolated from the symbiotic bacterium Fascaplysinopsis sp. of Fiji, and it has the function of promoting neurite growth and regeneration.
[0026] The molecular formula of 9-Methylfascaplysin is C 18 H 11 ClN2O, hereinafter referred to as 9-MF, has the following structural formula:
[0027]
[0028] This invention has found that 9-MF can promote neurite growth and regeneration, specifically by promoting the growth of PC12 cell neurites ( Figure 1 Increased expression of the growth-related protein GAP-43 ( Figure 2 ), the number of mitochondria increases ( Figure 3 The effect was similar to that of the positive control drug retinoic acid.
[0029] This invention, through transcriptome sequencing, identified 715 differentially expressed genes (DEGs) in PC12 cells after the addition of 9-MF, of which 224 genes were upregulated and 491 genes were downregulated. Figure 4 GO cellular component (GO-CC) analysis showed that 9-MF-associated DEGs were enriched in mitochondria and synaptic structures. Figure 5 KEGG pathway analysis showed that these genes are enriched in pathways of various neurodegenerative diseases and nervous system disorders. Figure 6 A protein interaction network was constructed using the STRING database and analyzed using Cytoscape software. Hub genes, including Rock2, Ppp1ca, Tuba1a, Tgfb2, Fos, ND1, ND2, ND5, Hras, Foxo3, and Irs1, were identified. The interaction network showed that Rock2 was located at the core. Figure 7 Venn diagram analysis showed that 339 genes were simultaneously present in 9-MF-related DEGs and gene sets related to neurite growth and regeneration. Figure 8GO molecular function (GO-MF) analysis of these genes revealed enrichment in kinase activity and microtubule binding function. Figure 9 ).
[0030] This invention uses the CMap database to predict the target of 9-MF using 9-MF-related DEGs. CMap analysis shows that 9-MF is positively correlated with the following factors: LIMK2 knockout, RhoGEF knockout, RhoGAPs overexpression, and ROCK2 inhibitor treatment; while it is negatively correlated with the following factors: RhoGEFs overexpression and RhoGAPs knockout. Figure 10 ROCK2 can directly phosphorylate LIMK; ROCK2 activity can be inhibited by Rho GTPase-activating proteins and promoted by Rho guanylate exchange factor, therefore 9-MF may act on ROCK2 and its related pathways.
[0031] This invention employs molecular docking technology to evaluate the binding affinity between ROCK2 and different inhibitors. The results show that the binding energies of seven known ROCK2 inhibitors (zerasudine, berumodine, hydroxyfasudil, lipasudine, azaindole-1, fasudil, and Y-27632) to ROCK2 are -9.41, -9.38, -8.22, -8.06, -7.84, -7.81, and -7.07 kcal / mol, respectively. Figure 11 Under the same conditions, 9-MF exhibited the best binding affinity (-10.0 kcal / mol). Optimal docking conformation analysis showed that 9-MF formed van der Waals interactions with residues of ROCK2, including Ile98, Gly99, Arg100, Gly101, Lys121, Val153, Glu170, Tyr171, Asp232, and Phe384, and hydrophobic interactions with residues of Ala119, Met172, and Ala231. Furthermore, 9-MF also exhibited π-σ interactions with Val106 and Leu221, and formed a π-sulfur bond contact with Met169. Figure 12 ).
[0032] This invention found that 9-MF can significantly reduce the area of pROCK2-positive regions in PC12 cells, and that 10 nM 9-MF has an inhibitory effect on ROCK2 comparable to that of 5 μM fasudil, a classic ROCK2 inhibitor. Figure 13 Western blot analysis confirmed that 9-MF can inhibit ROCK2 phosphorylation levels ( ). Figure 13 The ROCK2 activator narcissin can effectively antagonize the effect of 9-MF in promoting the growth and regeneration of PC12 cell neurites. Figure 14 ).
[0033] The following are the specific experimental methods used in this application.
[0034] PC12 cells are a recognized model for evaluating the ability of drugs to promote neurite growth and regeneration.
[0035] PC12 cells were cultured in Durbeco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin, and cultured at 37°C and 5% CO2. The medium was changed every two days. When the cell density reached 80-90%, the cells were seeded into 6-well or 96-well plates.
[0036] After cell adhesion and growth, 9-MF and a reference drug were added for comparison with the solvent control group. Neural neurite analysis was performed using FDA staining. Cell imaging was performed using a high-content ImageXPress Micro XL system with a 20x objective lens for image acquisition. FDA fluorescence signals were detected using a FITC filter, and image processing and analysis were performed using the "Neurite Outgrowth" module of MetaXpress software. Mitochondrial staining was performed using MitoTracker™ Green FM fluorescent dye. After incubating cells and dye at 37°C and 5% CO2 for 30 minutes, cells were washed three times with pre-warmed PBS buffer, and nuclear staining was performed with 4',6-diamidinyl-2-phenylindole (DAPI).
[0037] Cell imaging was performed using a high-content ImageXPress Micro XL system with a 20x objective lens for image acquisition. MitoTracker™ Green FM fluorescence signals were detected using a FITC filter, while DAPI fluorescence signals were acquired using a dedicated DAPI filter. All image processing and analysis were performed using a custom module of the MetaXpress software. The expression level of the growth-associated protein GAP-43 was assessed using immunofluorescence and Western blotting methods.
[0038] This invention uses TRIzol reagent to homogenize PC12 cells from the 9-MF treatment group and the solvent control group at 4°C for RNA sequencing. Genes with a p-value less than 0.05 and an absolute log2FC value greater than 0.3 were selected as differentially expressed genes. Human genome sequences and gene annotation information were obtained from the University of California, Santa Cruz Genome website (http: / / genome.ucsc.edu / ).
[0039] This invention retrieved 7629 genes related to neurite growth and regeneration and 11343 genes related to mitochondrial metabolism from the GeneCards database (https: / / www.genecards.org / ). All genes were uniformly converted to rat (Rattus norvegicus) homologs using the g:Profiler online tool (https: / / biit.cs.ut.ee / gprofiler / gost). Gene ontology enrichment analysis and pathway enrichment analysis were performed using the David Bioinformatics Resource Platform (https: / / David.ncifcrf.gov). Bubble charts were generated using the Bioinformatics Online website (https: / / www.bioinformatics.com.cn / ). Protein-protein interaction network analysis was performed using the STRING online database and visualized using Cytoscape software (version 3.10.3).
[0040] This invention selects 150 genes each of the most significantly upregulated and downregulated genes from transcriptome data for comparative analysis in the CMap database to screen potential small molecule regulators and therapeutic candidates. This analysis covers the characteristic profiles of 8559 perturbation factors, systematically evaluating the association between the target gene set and the perturbation factors in the entire CMap library. The CMap score ranges from -99.39 to 99.82; a higher score indicates a more significant regulatory similarity between the compound and the target gene set.
[0041] This invention obtained the high-resolution crystal structure (PDB ID: 6ED6) of the ROCK2 protein by screening from the RCSB PDB database (http: / / www.rcsb.org / ). The chemical structure of 9-MF was processed using PyMOL software, and molecular docking was performed using AutoDockVina 1.5.6 to study protein-ligand interactions. The coordinates of the docking grid were set to cover the entire binding site region, and the optimal binding conformation was selected based on the docking score. Finally, protein-ligand interactions were visualized using PyMOL.
[0042] This invention evaluates the expression level of p-ROCK using cell immunofluorescence and Western blotting methods.
[0043] As can be seen from the above description, the 9-MF in this invention can promote the growth and regeneration of neurites and can be used to treat neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as various neurological diseases such as ischemic stroke, hemorrhagic stroke, and traumatic brain injury.
[0044] The scope of protection of this invention includes, but is not limited to, the above embodiments. The scope of protection of this invention is defined by the claims. Any substitutions, modifications, or improvements to this technology that are easily conceived by those skilled in the art fall within the scope of protection of this invention.
Claims
1. Use of 9-methylfascaplysin for the manufacture of a medicament for the treatment or prevention of a disease of the nervous system, characterized in that, The medicine includes one of tablets, capsules, pills, injections, oral agents, suspensions containing 9-Methylfascaplysin.
2. The use of 9-methylfascaplysin according to claim 1 for the preparation of a medicament for the treatment or prevention of a nervous system disease, characterized in that, The nervous system disease includes at least one of Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, ischemic stroke, hemorrhagic stroke, traumatic brain injury.