Application of BC1618 in preparation of medicine for protecting central nervous system

Abstract

The invention provides application of BC1618 in preparation of a central nervous system protection medicine, and belongs to the technical field of medicine. As a novel drug, the BC1618 inhibits a key component subunit FBXO48 of E3 ubiquitin ligase and further stabilizes SERBP1 so as to play a nerve protection function. The research of the invention shows that the BC1618-FBXO48-SERBP1 axis can promote the survival of retinal ganglion cells and the regeneration of axons after optic nerve injury, and delay the degeneration of axons at the same time. In addition, the nerve protection function of the BC1618 can be further applied to a Parkinson's disease model. Therefore, the BC1618 can be used as a potential medicine composition for preventing or treating and controlling nervous system development disorder related diseases, and can remarkably improve the symptoms such as neuron death, axon loss, neuron dysfunction and dyskinesia caused by central nervous system injury and diseases (such as optic nerve injury and Parkinson's disease).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of BC1618 in the preparation of neuroprotective drugs for the central nervous system. Background Technology

[0002] Neurological diseases affect more than 40% of the global population, causing irreversible neuronal damage and disruption of neural circuits, resulting in a devastating socioeconomic burden (Steinmetz et al., 2021). Neuronal death and axonal loss are common pathological features of various central nervous system (CNS) diseases (Gómez-Deza et al., 2024), including acute injuries (such as optic nerve injury ONC, traumatic brain injury TBI) (Tran et al., 2019; Sivandzade et al., 2020), neurodegenerative diseases (such as Alzheimer's disease AD, Parkinson's disease PD), and other diseases (Canter et al., 2016; Castonguay et al., 2021; Ghanem et al., 2024; Margolis et al., 2015).

[0003] Over the past few decades, advancements in medicine, surgery, and rehabilitation have led to significant scientific progress and substantial breakthroughs in improving neuronal survival and axonal regeneration after CNS injury (Sujin et al., 2019; Park et al., 2008; Lu et al., 2020; Huang et al., 2021; Li et al., 2023). However, the number of surviving neurons and the length of axonal regeneration still need improvement, and key gaps in our understanding of neuroprotection and axonal regeneration in CNS diseases remain. Therefore, developing effective pharmacological interventions and elucidating the mechanisms by which neuronal survival and long-distance axonal regeneration are crucial frontiers of treatment with profound clinical significance.

[0004] The ubiquitin signaling pathway—an evolutionarily conserved pathway from invertebrates to mammals—plays a central role in regulating protein homeostasis in CNS diseases (Bett et al., 2016). Neuronal ubiquitin homeostasis is crucial for protein homeostasis and functional plasticity, and its dysregulation can lead to neurological disorders through impaired clearance of misfolded / aggregated proteins (Henneberg et al., 2021; Zenge et al., 2024). The localization of ubiquitin signaling in neuronal subdomains highlights its regulatory complexity, and its dysregulation in disease reveals the therapeutic potential for neuroprotection through the regulation of these pathways. Growing evidence suggests that ubiquitin system regulators, particularly E3 ligases and deubiquitinating enzymes (DUBs), are key regulators of neuronal survival pathways in CNS diseases (Schmidt et al., 2021; Guerroué et al., 2020; Kumar et al., 2020). Therefore, targeting ubiquitin system molecules is a promising therapeutic strategy for CNS repair. However, systematic screening of these molecules for CNS damage repair remains insufficient, and a large number of untapped therapeutic targets have not yet received attention. Summary of the Invention

[0005] Through in-depth research, the inventors discovered therapeutic small molecule compounds for central nervous system injury or neurodegenerative diseases, and further confirmed the therapeutic efficacy and concentration-dependent nature of these therapeutic agents. These agents significantly improve symptoms such as neuronal death and axonal degeneration caused by central nervous system injury, or dopaminergic neuronal death, motor dysfunction, and neurodegenerative lesions caused by neurodegenerative diseases (e.g., Parkinson's disease). The purpose of this invention is to provide a method for preventing and / or treating at least one symptom or pathological feature of central nervous system injury or neurodegenerative diseases in subjects, the method comprising: administering to a subject in need a small molecule compound BC1618 capable of modulating E3 ubiquitin ligase activity (e.g., FBXO48), the drug being used to prevent and treat at least one symptom or pathological feature of neurodegenerative diseases or central nervous system injury in subjects. This invention also provides the use of a therapeutic agent capable of modulating changes in modification levels caused by E3 ubiquitin ligase activity (e.g., FBXO48) in the preparation of a medicament for preventing and treating at least one symptom or pathological feature of neurodegenerative diseases or central nervous system injury in subjects.

[0006] In some embodiments, at least one symptom or pathological manifestation of the central nervous system injury and disease is selected from neuronal survival, axonal regeneration and degeneration, maintenance of neural circuits, and restoration of motor function.

[0007] In some embodiments, the drug can improve at least one symptom or pathological manifestation of central nervous system injury and disease, such as neuronal survival, axonal regeneration and degeneration, maintenance of neural circuits, and restoration of motor function.

[0008] In some implementations, the central nervous system injury and disease is selected from optic nerve injury, spinal cord injury, stroke, Alzheimer's disease, Parkinson's disease, etc.

[0009] In some implementations, the subject is a mammal.

[0010] In this article, "gene expression" refers to the process by which the information contained in a gene is transformed into a gene product. Gene products can be direct transcription products of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNA, shRNA, RNAi, miRNA, or any other type of RNA) or proteins produced by the translation of mRNA. Gene products also include modified RNA and modified proteins. RNA modification processes include capping, polyadenylation, methylation, and editing; protein modification processes include methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, tetradecylation, and glycosylation. "Regulation" of gene expression refers to changes in gene activity, which may include, but is not limited to, gene activation and gene repression.

[0011] In some embodiments, the reagent described herein, BC1618 (its pharmaceutically acceptable salts or esters, prodrugs, stereoisomers, hydrates, solvates, crystal forms, their metabolite forms, or any combination or mixture thereof), may be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions and lyophilized powders), inhalers, sprays, etc. It may be present in the pharmaceutical composition in unit dose form to facilitate administration by any suitable method known in the art, including but not limited to oral, rectal, parenteral, or topical administration.

[0012] In some implementations, BC1618 described herein is an orally active Fbxo48 inhibitor that can regulate gene expression changes by increasing the stability of the SERBP1 protein.

[0013] In addition, other carrier materials and routes of administration known in the pharmaceutical field may be used. BC1618, as described herein, can be prepared using any well-known pharmaceutical process, such as effective formulations and routes of administration. The above considerations regarding effective formulations and routes of administration are well-known in the art and described in standard textbooks. Formulations of the drug are described, for example, in Hoover, John E., Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd edition), American Pharmaceutical Association, Washington, 1999.

[0014] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution: the application of BC1618 in the preparation of drugs for the protection of the central nervous system, wherein the small molecule compound BC1618 is used to prepare drugs for the prevention or treatment of central nervous system damage or neurodegenerative diseases.

[0015] Preferably, the central nervous system injury or neurodegenerative disease is selected from optic nerve injury, spinal cord injury, stroke, Alzheimer's disease or Parkinson's disease.

[0016] Preferably, the drug exerts its neuroprotective effect by inhibiting the E3 ubiquitin ligase activity of FBXO48.

[0017] Preferably, inhibiting the E3 ubiquitin ligase activity of FBXO48 leads to increased protein stability of its substrate SERBP1.

[0018] The FBXO48 catalyzes the K63-linked ubiquitination of lysine (K52) at position 52 of the SERBP1 protein.

[0019] Preferably, the preventive or therapeutic effects of the drug include at least one of the following: (a) improving the survival rate of damaged neurons; (b) promoting the regeneration of damaged neuronal axons; (c) delaying the degeneration of damaged neuronal axons; (d) maintaining or restoring neural circuit function; and (e) improving motor dysfunction.

[0020] Preferably, the drug is used to treat optic nerve injury, and its effects include improving the survival rate of retinal ganglion cells (RGCs) and promoting their axonal regeneration.

[0021] Preferably, the drug is used to treat Parkinson's disease, and its effects include protecting the survival of dopaminergic neurons in the substantia nigra pars compacta, preventing the loss of striatal dopaminergic fibers, and improving motor behavior deficits.

[0022] To achieve the objectives of this invention, another technical solution provided by this invention is: a pharmaceutical composition comprising a therapeutically effective amount of a small molecule compound BC1618, a pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, or crystal form thereof, and one or more pharmaceutically acceptable carriers or excipients. Its dosage form is a tablet, pill, suspension, emulsion, solution, gel, capsule, powder, granule, elixir, lozenge, suppository, injection, inhaler, or spray.

[0023] Compared with the prior art, the present invention achieves the following technical effects: This invention has discovered therapeutic targets for central nervous system injuries and diseases, and further obtained a class of drugs targeting the above targets, which can significantly improve symptoms such as neuronal death, axonal loss, neuronal dysfunction, and motor disorders caused by central nervous system injuries and diseases (such as optic nerve damage and Parkinson's disease). Attached Figure Description

[0024] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0025] Figure 1 This is a diagram illustrating the neuroprotective and neurite-promoting effects of compound BC1618 in an in vitro model. (Figure:) A represents the quantitative HT22 cell viability after 48 hours of glutamate stimulation in Example 1. The scatter plot data is the mean ± standard error. p < 0.05.

[0026] B represents the timeline for screening hippocampal neurons in Example 1.

[0027] Image C shows representative images of surviving hippocampal neurons in different compound treatment groups in Example 1, labeled with neuron-specific markers MAP2 (green), NeuN (red), and DAPI (nuclear staining, blue). Scale bar: 200 µm.

[0028] D represents the quantitative survival rate of hippocampal neurons in the compound-treated group in Example 1. (n = 6 independent experiments, data are mean ± standard error. Unpaired two-tailed t-test, *) p < 0.05, ** p < 0.01, *** p < 0.001).

[0029] E represents the quantitative survival rate of hippocampal neurons in the BC1618-treated group in Example 1. BC1618 promotes neuronal survival in a concentration-dependent manner (n = 3 independent experiments; data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparison test, ns = no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0030] F represents a representative neuron image from Example 1, taken at DIV3 after 48 hours of treatment with DMSO or different concentrations of BC1618, and is marked with MAP2 (green). Scale bar: 50 µm.

[0031] G represents the quantification of the total neurite length of hippocampal neurons in Example 1. 2 µM BC1618 significantly increased the maximum total neurite length (n = 3 independent experiments; n in the figure represents the number of cells analyzed; data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparison test, ns = no significant difference, * p < 0.05, *** p < 0.001).

[0032] H represents the quantification of the longest neurite length in hippocampal neurons in Example 1. 2 µM BC1618 significantly increased the longest neurite length (n = 3 independent experiments; n in the figure represents the number of cells analyzed; data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparison test, ns = no significant result, * p < 0.05, *** p < 0.001).

[0033] Figure 2 This is an in vivo study of BC1618 promoting retinal ganglion cell survival and axonal regeneration in a optic nerve injury model. (Figure:) A represents the timeline of the treatment regimen for BC1618 (1-20 mM) after optic nerve injury in Example 2.

[0034] Image B shows representative images of RBPMS (green)-marked RGCs in each group of retinal full-slice patches 2 weeks after optic nerve injury in Example 2. Scale bar: 50 µm.

[0035] C represents the quantification of RGC survival rate 2 weeks after injury in Example 2. The 2 mM BC1618 group had the highest RGC survival rate (n = 4 animals, data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparison test, ns = no significant difference, * p < 0.05, *** p < 0.001).

[0036] D represents CTB-labeled axons of the optic nerve in mice treated with DMSO or BC1618 (1-20 mM) in Example 2. White lines indicate lesion sites, and insets show regenerated axons at distances of 500, 1000, or 1500 µm from the lesion site. Scale bar: 100 µm.

[0037] E represents the quantitative analysis of regenerated axons at different distances distal to the injury site in Example 2 (n = 6 animals, data are mean ± standard error). One-way ANOVA was followed by Tukey's multiple comparison test. * p < 0.05, ** p < 0.01, *** p <0.001).

[0038] Figure 3 The figure shows the dose-dependent safety study of BC1618 on retinal ganglion cells, where: Image A shows representative images of RBPMS (green) labeled retinal ganglion cells (RGCs) in each group of retinal whole-slice retina one week after BC1618 injection in Example 2. Scale bar: 50 µm.

[0039] B represents the quantitative analysis of RGC survival rate one week after BC1618 injection in Example 2. Except for 20 mM, BC1618 did not affect RGC survival at any of the tested concentrations (n=3 animals, data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparison test, ns= no significant difference. p < 0.01).

[0040] Figure 4 This is a diagram validating the dual role of BC1618 in delaying axonal degeneration and promoting regeneration after optic nerve injury. (The diagram shows...) A represents the timeline of the BC1618 treatment regimen in Example 2. Six-week-old mice were first injected with CTB-488, then injected with DMSO or BC1618 two days after optic nerve injury, and then injected with CTB-555 one day later. Phenotypes were observed three days after injury.

[0041] B represents the CTB-marked axons of the optic nerve in the DMSO / BC1618 treatment group in Example 2. White lines indicate lesion sites, and arrows point to regenerating axons. Scale bar: 100 µm.

[0042] Figure 5 Representative images of retinal sections from Example 2, showing the inflammatory response of BC1618: TUJ1 (red), CD68 (green), IBA1 (white), DAPI (nuclear staining, blue). Scale bar: 50 µm.

[0043] Figure 6 Figure 1 shows the long-term protective effect of Fbxo48 knockdown on retinal ganglion cells and its axonal regeneration effect. A represents the timeline of the AAV2 virus treatment scheme in Example 3.

[0044] Image B is a representative image of the RBPMS (green)-marked RGC on the retinal pannus at a prolonged time point after optic nerve injury in Example 3. Scale bar: 50 µm.

[0045] C represents AAV2- in Example 3. control or AAV2- Fbxo48-shRNA CTB markers were used to label axons of the optic nerve in the treated group. White lines indicate lesion sites, and insets show regenerated axons at 500 or 1000 µm from the lesion site. Scale bar: 100 µm.

[0046] D represents the quantification of RGC survival rate from 2 weeks to 6 months post-injury in Example 3. AAV2- Fbxo48-shRNA The group showed long-term protective effects against RGC (n=3-4 animals, data are mean ± standard error. Unpaired two-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0047] E represents the quantitative analysis of regenerated axons at different distances distal to the injury site in Example 3 (n=6 animals, data are mean ± standard error. Unpaired two-tailed t-test, ns= no significant, * p < 0.05, *** p < 0.001).

[0048] Figure 7 Figure 1 shows the mechanism by which Fbxo48 overexpression antagonizes BC1618-mediated retinal ganglion cell protection and axonal regeneration. A is AAV2- in Example 3. Fbxo48 Timeline of the joint processing solution with BC1618.

[0049] Image B is a representative image of the RBPMS (green)-marked RGC in a full-slice retinal patch after optic nerve injury in Example 3. Scale bar: 50 µm.

[0050] C represents the quantification of RGC survival rate 2 weeks after injury in Example 3. Overexpression Fbxo48 Complete elimination of the protective effect of BC1618 on RGCs (n=5-7 animals, data are mean ± standard error). Tukey's multiple comparison test after one-way ANOVA showed no significant difference (ns=). p < 0.001).

[0051] D represents CTB-labeled axons of the optic nerve in different treatment groups in Example 3. White lines indicate lesion sites, and insets show regenerated axons at distances of 500, 1000, or 1500 µm from the lesion site. Scale bar: 100 µm.

[0052] E represents the quantitative analysis of regenerated axons at different distances distal to the lesion site in Example 3. Overexpression Fbxo48 Complete elimination of the promoting effect of BC1618 on axonal regeneration (n=6 animals, data are mean ± standard error. Tukey multiple comparison test after one-way ANOVA, ns= no significant, *) p < 0.05, ** p < 0.01, *** p < 0.001).

[0053] Figure 8 The figure shows the validation of the ineffectiveness of the AMPK activator AICAR in optic nerve injury repair and the study on the regulation of SERBP1 protein levels by Fbxo48. A represents the timeline of the AICAR processing scheme in Example 4.

[0054] Image B is a representative image of the RBPMS (green)-marked RGC in a full-slice retinal patch after optic nerve injury in Example 4. Scale bar: 50 µm.

[0055] C represents the quantification of RGC survival rate 2 weeks after injury in Example 4. AICAR injection had no protective effect on RGC (n=5 animals, data are mean ± standard error. Unpaired two-tailed t-test, ns= no significance).

[0056] D represents the CTB-marked axons of the optic nerve in the DMSO or AICAR treatment group in Example 4. White lines indicate the lesion site; the inset shows the regenerated axon at 500 µm from the lesion site. Scale bar: 100 µm.

[0057] E represents the quantitative analysis of regenerated axons at different distances distal to the injury site in Example 4. AICAR injection did not promote axonal regeneration (n=5-6 animals, data are mean ± standard error. Tukey multiple comparison test after one-way ANOVA, ns= no significance).

[0058] F represents AAV2- in Example 4. Fbxo48-sh and AAV2- Fbxo48 Representative images of SERBP1 changes in RGC 14 days after viral injection: RBPMS (magenta), SERBP1 (green), DAPI (blue). Scale bar: 50 µm.

[0059] G is in Example 4 Figure 8 Quantitative analysis of SERBP1 fluorescence intensity in RGCs of F animals (n=3 animals, n value in the figure is the number of cells analyzed, data are mean ± standard error. Unpaired two-tailed t-test, *** p < 0.001).

[0060] Figure 9 The figure shows the identification of SERBP1 as a key downstream effector of FBXO48 and its function in optic nerve injury repair. A shows the LC-MS proteomic volcano plot from Example 4. 1640 differentially expressed proteins (BC1618 vs. DMSO) are labeled as upregulated (pink) and downregulated (green). P The values ​​were calculated using a two-tailed unpaired t-test.

[0061] B is a Western blot analysis from Example 4, which showed that SERBP1 protein levels in HT22 cells increased after 1 day of treatment with 10 µM BC1618.

[0062] C represents the SERBP1 protein level quantification in Figure (B) of Example 4 (n=3 independent experiments, data are mean ± standard error. Unpaired two-tailed t-test, * p < 0.05).

[0063] D shows a retinal section from Example 4, illustrating how BC1618 protects SERBP1 in the RGC from degradation 7 days after injection. The sections were stained with RBPMS (magenta), SERBP1 (green), and DAPI (blue). Scale bar: 50 μm.

[0064] E represents the quantitative analysis of SERBP1 fluorescence intensity in RGCs (D) of Example 4 (n=3 animals, where n is the number of cells analyzed, and data are mean ± standard error). Unpaired two-tailed t-test was used. * p < 0.001).

[0065] F is a representative image of the RBPMS (green)-marked RGC on a full retinal patch two weeks after optic nerve injury in Example 4. Scale bar: 50 µm.

[0066] G represents the quantification of RGC survival rate 2 weeks after injury in Example 4. AAV2- Serbp1 The group significantly improved RGC survival rate (n=4 animals, data are mean ± standard error). Unpaired two-tailed t-test, * p < 0.001).

[0067] H represents AAV2- in Example 4. control or AAV2- Serbp1 CTB markers were used to label axons of the optic nerve in the treated group. White lines indicate lesion sites, and insets show regenerated axons at 500 or 1000 µm from the lesion site. Scale bar: 100 µm.

[0068] I represents the quantitative analysis of regenerated axons at different distances distal to the lesion site in Example 4. AAV- Serbp1 The group exhibited axonal regeneration capacity 14 days after optic nerve injury (n=6 animals, data are mean ± standard error). After one-way ANOVA and Tukey's multiple comparisons test, ns= no significant result was observed. * p < 0.05, ** p < 0.01, *** p < 0.001).

[0069] Figure 10 Figure 1 shows a study on the partial elimination of BC1618-mediated retinal ganglion cell protection by knocking down Serbp1 and its complete antagonism of axonal regeneration. a is AAV2- in Example 5 Serbp1-shRNA Timeline of the joint processing solution with BC1618.

[0070] b is a representative image of the RBPMS (green)-marked RGC in the ONC posterior retinal full-coverage patch of Example 5. Scale bar: 50 µm.

[0071] c represents the quantification of RGC survival rate 2 weeks after injury in Example 5. Knockdown Serbp1 Partial elimination of the protective effect of BC1618 on RGCs (n = 4 animals, data are mean ± standard error). Tukey multiple comparison test followed by one-way ANOVA. p < 0.01, *** p < 0.001).

[0072] d represents CTB-labeled axons of the optic nerve in different treatment groups in Example 5. White lines indicate lesion sites, and insets show regenerated axons at distances of 500, 1000, or 1500 µm from the lesion site. Scale bar: 100 µm.

[0073] e represents the quantitative analysis of regenerated axons at different distances distal to the lesion site in Example 5. Knockdown Serbp1 Complete elimination of the promoting effect of BC1618 on axonal regeneration (n = 6 animals, data are mean ± standard error. Tukey multiple comparison test after one-way ANOVA, ns = no significant difference, *) p < 0.05, *** p < 0.001).

[0074] Figure 11 The diagram illustrates the mechanism by which combined knockdown of Fbxo48 and Serbp1 protects retinal ganglion cells and promotes axonal regeneration. (Figure:) A is AAV2- in Example 5. Fbxo48 shRNA With AAV2- Serbp1 shRNA Timeline of the joint processing solution.

[0075] Image B is a representative image of the RBPMS (green)-marked RGC in a full-slice retinal patch after optic nerve injury in Example 5. Scale bar: 50 µm.

[0076] C represents the quantification of RGC survival rate 2 weeks after injury in Example 5. Knockdown Serbp1 Partial elimination of AAV2- Fbxo48 shRNA Protective effect against RGC (n = 5 animals, data are mean ± standard error). One-way ANOVA followed by Tukey multiple comparison test, *** p < 0.001).

[0077] D represents CTB-labeled axons of the optic nerve in different treatment groups in Example 5. White lines indicate lesion sites, and insets show regenerated axons at 500 or 1000 µm from the lesion site. Scale bar: 100 µm.

[0078] E represents the quantitative analysis of regenerated axons at different distances distal to the lesion site in Example 5. Knockdown Serbp1 Completely eliminate AAV2- Fbxo48 shRNA The promoting effect on axonal regeneration (n = 6 animals, data are mean ± standard error. After one-way ANOVA and Tukey's multiple comparison test, ns = no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0079] Figure 12 This diagram illustrates the molecular mechanism of the interaction between FBXO48 and SERBP1 and the K63-linked ubiquitination modification. (Figure:) A represents the immunoprecipitation analysis of the interaction between FBXO48 and SERBP1 in HEK293T cells in Example 6. SERBP1 was immunoprecipitated with anti-FLAG (FBXO48) antibody.

[0080] B represents the immunoprecipitation analysis of the interaction between SERBP1 and FBXO48 in HEK293T cells in Example 6. Anti-SERBP1 antibody immunoprecipitation of FLAG (FBXO48).

[0081] C represents 293T cells co-transfected with Serbp1-His and WT Ub-HA plasmids as described in Example 6, along with an empty vector control or Fbxo48-Flag plasmid. After treatment with 10 μM MG132 for 6 hours, the cells were collected for ubiquitination detection.

[0082] D represents 293T cells co-transfected with Fbxo48-Flag and Serbp1-His plasmids as described in Example 6. After treatment with WTUb-HA or different Ub-HA mutants (K48R, K63R) for 6 hours, the cells were collected for ubiquitination detection.

[0083] E represents 293T cells transfected with Fbxo48-Flag and WT Ub-HA plasmids as described in Example 6. These cells were treated with 10 μM MG132 for 6 hours using WTSerbp1-His or different Serbp1-His mutants (K32R, K52R, K68R, K122R) for ubiquitination detection. (AE) Representative immunoblots were obtained from three independent biological experiments.

[0084] Figure 13 Figure 1 shows a study on the protection of dopaminergic neurons and improvement of motor dysfunction by oral administration of BC1618 in an MPTP-induced Parkinson's disease model. A represents the timeline of Parkinson's disease treatment in Example 7.

[0085] B shows a representative TH immunofluorescence section of the striatum and substantia nigra pars compacta (SNpc) of an 8-week-old male mouse in Example 7. The inset shows a magnified image of a surviving dopaminergic neuron. Scale bar: 250 µm.

[0086] C represents the quantification of striatal TH⁺ dopaminergic fibers in Example 7. Oral administration of BC1618 prevented TH⁺ fiber loss (n = 4 animals; data are mean ± standard error). Unpaired two-tailed t-test, ns = no significant difference. p < 0.01, *** p < 0.001).

[0087] D represents the quantitative measurement of TH⁺ cell survival in the SNpc region in Example 7. Oral administration of BC1618 prevented the loss of TH⁺ dopaminergic cells (n = 3 animals, data are mean ± standard error). Unpaired two-tailed t-test, ns = no significant, * p <0.05).

[0088] E and F are the quantitative results of the pole climbing test (E) and gripping test (F) in Example 7. Oral administration of BC1618 improved MPTP-induced behavioral deficits (n = 7-10 animals, data are mean ± standard error. Unpaired two-tailed t-test, ns = no significance, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0089] G is a schematic diagram of the directed differentiation of midbrain dopamine neurons in Example 7.

[0090] H is a representative image of TH immunofluorescence in midbrain dopamine neurons from Example 7. Scale bar: 50µm I represents the quantification of TH⁺ midbrain dopamine neurons in Example 7. BC1618 treatment prevented the loss of TH⁺ midbrain dopamine neurons (n=3 independent biological experiments, data are mean ± SEM. Unpaired two-tailed t-test, ns= no significant, *). p < 0.05).

[0091] Figure 14 This is an overview of research on the multimodal neuroprotective and regenerative mechanisms of BC1618 in neurodegenerative diseases. Detailed Implementation

[0092] The following are specific embodiments of the present invention, in conjunction with the appendix. Figure 1-14 The technical solutions of the present invention will be further described below, but the present invention is not limited to these embodiments; in the following description, specific details such as specific configurations are provided only to help to fully understand the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0093] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0094] Unless otherwise specified, the materials, practices, and experimental equipment involved in the embodiments of this invention are all commercially available products in the relevant chemical and biotechnology fields.

[0095] 1. Cell viability assay HT22 cell line (Procell, CL-0595) was cultured in DMEM (Gibco) medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Procell, PB180120). Cells were cultured in a humidified environment at 37°C and 5% CO2. For stimulation, HT22 cells were seeded in 96-well plates (5 × 10³ cells per well) and cultured for 24 hours. Cells were then treated with different concentrations of glutamate (Sigma, G5889) for 48 hours. In the treatment groups, the drug was added simultaneously with glutamate. Cell viability was determined by measuring absorbance (450 nm) after adding Enhanced Cell Counting Kit-8 (CCK-8) (Beyotime, C0042).

[0096] 2. Primary hippocampal neuron culture Primary hippocampal neurons were obtained from wild-type mice at day 0 (P0). Briefly, mouse brains were removed and hippocampal tissue was isolated in ice-cold PBS. After trypsin digestion for 10 minutes, the tissue was gently pipetted and allowed to stand for a short time. The supernatant was collected for cell counting. The isolated hippocampal neurons were seeded in poly-D-lysine (PDL; Sigma-Aldrich, P6407)-coated glass-bottomed culture dishes (for neurite growth analysis) or 96-well plates (for high-content screening) and cultured in neurobasal medium (Gibco, 21103049) supplemented with 2% B-27 (Gibco, 12587010), 1% GlutaMAX (Gibco, 35050061), and 1% penicillin-streptomycin. Neurons were cultured for 10 days in a humidified environment at 37°C and 5% CO2. For neurite growth analysis, ImageJ software was used to analyze the total length and longest neurite length of individual neurons. Fluorescence signals were segmented using a thresholding tool, and the fluorescence images were converted to grayscale to trace intact cells. All branches emanating from the cell body were identified and counted. For high-content screening, nine fields of view were taken from the central region of each well in a 96-well plate, covering almost the entire well. All groups were then automatically counted using the same thresholding settings.

[0097] 3. Immunostaining Hippocampal neurons were fixed with 4% paraformaldehyde for 15 minutes, then thoroughly washed and stored in PBS at 4°C. Cells were blocked in PBS blocking buffer containing 2% BSA and 0.3% Triton X-100 at room temperature for 1 hour. They were then incubated overnight at 4°C with primary antibodies: mouse anti-MAP2 (Sigma-Aldrich, MAB3418, 1:500) and rabbit anti-NeuN (Abcam, ab177487, 1:500). For tissues, mice were anesthetized, perfused with PBS via the heart, and then perfused with 4% PFA. The tissues were then post-fixed overnight in 4% PFA. For sections, the retina and brain were immersed in 30% sucrose for 24–48 hours for cryoprotection. 20 μm thick retinal sections and 40 μm thick brain sections were obtained using a Leica CM1950 cryostat or a Leica SM2010 R sliding microtome. Sections were stored at -20°C. Retinal sections were washed with PBS, blocked in PBS containing 2% BSA and 0.3% Triton X-100 at room temperature for 1 hour, and then incubated overnight at 4°C with primary antibodies: guinea pig anti-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), mouse anti-SERBP1 (Proteintech, 67792-1-Ig, 1:500), and rabbit anti-FBXO48 (Biorbyt, orb183658, 1:500). For whole retinal slices, the retina was radially cut into petal shapes, washed three times with PBS, blocked in PBS containing 5% donkey serum and 0.5% Triton X-100 at room temperature for 1-2 hours, and then incubated with primary antibody at 4°C for 2 days: rabbit anti-RBPMS (Abcam, ab194213, 1:300). Brain sections were post-fixed in 4% PFA for 15 minutes, then washed three times with PBS. Next, the sections were permeabilized in 0.5% Triton X-100 for 20 minutes, and then blocked in PBS containing 2% BSA and 0.3% Triton X-100 at room temperature for 1 hour. They were then incubated overnight at 4°C with primary antibody: rabbit anti-tyrosine hydroxylase (Cell Signaling Technology, 58844, 1:500). All immunostained samples were washed three times with PBS for 10 minutes each time.Incubate with goat or donkey-derived secondary antibodies (Invitrogen, 1:500-1000) tagged with Alexa Fluor™ and matched to the host species of the primary antibody for 1-2 hours in a dark room at room temperature.

[0098] 4. Animals All animal experiments were approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences, and conducted in accordance with the National Guidelines for Animal Care and Use Ethics (IOZ-IACUC-2022-137). Primary hippocampal neurons were obtained from the dissection of wild-type male and female mice (C57BL / 6J, The Jackson Laboratory) at day 0 (P0) for culture. In all other experiments, wild-type male and female mice aged 5–8 weeks were used. All mice were housed in a specific pathogen-free (SPF) facility with a 12-hour light / dark cycle (lighting time: 7:00 AM to 7:00 PM). Food and water were freely available, and no sex-specific differences were observed.

[0099] 5. Intravitreal injection and optic nerve injury Intravitreal injections and optic nerve injury procedures are as described above³ 9 ⁻ 4 ¹. For AAV2-based experiments, mice were first anesthetized, and then 1–2 μL of AAV2 virus (5 × 10¹² GC / mL) was injected into the vitreous humor of the right eye using a glass micropipette connected to a microinjection pump. Care was taken to avoid damaging the lens and vortex veins. For optic nerve injury, mice were anesthetized, and an incision was made in the conjunctiva using microscissors to expose the right optic nerve. The nerve was clamped for 5 seconds approximately 0.5 mm posterior to the optic disc using Dumont #5 fine forceps. Immediately after injury, BC1618 or 10% DMSO was administered via intravitreal injection. Subsequently, mice received intravitreal injections of BC1618 or 10% DMSO weekly following optic nerve injury. To label retinal ganglion cell axons in the optic nerve, 1.0 μL of AlexaFluor 555-labeled cholera toxin B subunit (CTB) (1 μg / μL, Thermo Fisher Scientific) was administered using a pulled glass micropipette 2 days prior to perfusion. A small number of mice with macroscopic evidence of intraorbital infection were excluded from the analysis.

[0100] 6. Survival rate of retinal ganglion cells To quantify RGC survival, whole-retinal slices were immunostained with RBPMS antibodies to label surviving RGCs, following the steps outlined above (see Immunostaining). Six to eight fields of view were randomly selected from the peripheral region of each retina using a Zeiss LSM 880 microscope with a 20x objective lens. RGC survival was calculated by dividing the average number of RBPMS+ cells in the damaged retina (right) by the average number in the undamaged retina (left).

[0101] 7. Analysis of optic nerve tissue processing and regeneration The dehydration and transparency of the optic nerve are based on previous research³ 9 ⁻ 4 ¹The procedure was performed. In short, the optic nerve was first incubated in increasing concentrations of tetrahydrofuran (THF, Sigma-Aldrich) (50%, 70%, 80%, 100%, 100%). The optic nerve was then incubated in a benzyl benzoate / benzyl alcohol (BBBA, 2:1, Sigma-Aldrich) solution until completely transparent. The transparent optic nerve was mounted on a microscope slide with a clearing solution and stored at room temperature in the dark until imaging. For regeneration analysis, Z-axis stacked (step: 2 μm) and stitched fluorescence images were acquired using a Zeiss LSM 880 confocal microscope with 20x objectives. All images were subjected to maximum intensity projection for quantification. To quantify the number of regenerating axons in each optic nerve, a series of 10 μm thick optical sections were generated by maximum projection every five consecutive planes. The number of regenerating axons in all Z-projection images was counted and summed at intervals of 250 μm from the lesion site.

[0102] 8. Plasmid construction and site-directed mutagenesis The shRNA sequences of Fbxo48 and Serbp1 were cloned into the pAAV-U6-CAG-GFP vector. Mouse open reading frames (ORFs) of Fbxo48 and Serbp1 were amplified by PCR from mouse retinal cDNA. The Fbxo48 and Serbp1 ORFs with restriction enzyme sites were subcloned into the pAAV-EGFP (Addgene, 37825) AAV construct under the CAG promoter to obtain AAV2-CAG-Fbxo48 and AAV2-CAG-Serbp1, respectively. Eukaryotic expression plasmids containing the Serbp1 K32R-122R and Serbp1 K190R-222R mutants were synthesized using the Tsynth™ gene synthesis service. Mutant plasmids containing Fbxo48 3×Flag, Serbp1 K32R, Serbp1 K52R, Serbp1 K68R, and Serbp1 K122R were generated by PCR amplification. Primers used are listed in Table S1. All constructs were validated by DNA sequencing. The HA-UB (WT, K48R, K63R) (P69179, P69176, P69178) plasmids were purchased from MiaoLing Plasmid, China. The control AAV2-PLAP was purchased from Vigene Biosciences (Jinan, China). All AAVs were subsequently packaged by Delivectory Biosciences (Beijing, China) into serotype 2 / 2 AAVs (titer: 5 × 10¹² GC / mL).

[0103] 9. LC-MS Analysis HT22 cells were first cultured in 6-well plates to 70%-80% confluence. After treatment with 10 μM BC1618 or DMSO for 24 hours, cells were lysed for 30 minutes in RIPA lysis buffer (Beyotime Biotechnology, P0013B) supplemented with the protease inhibitor PMSF (Beyotime Biotechnology, ST506). The cell lysates were centrifuged at 13,000 rpm for 15 minutes at 4°C, and protein concentrations were determined using the BCA protein quantification kit (Beyotime Biotechnology, P0012). LC-MS / MS analysis was performed on the BC1618 and DMSO groups. Trypsin-digested peptides were analyzed on an Easy-nLC 1200 HPLC system (Thermo Fisher Scientific) coupled with an Orbitrap Exploris 480 (Thermo Fisher Scientific). MS data were acquired using the Orbitrap mass analyzer in data-dependent acquisition mode. Full-scan MS data were acquired at a high resolution of 120,000 (m / z 200) in the range of 350 to 1500 m / z. For peptide analysis, MS / MS data were acquired at a resolution of 15,000 (m / z 200). The resulting MS / MS data were processed and quantified using Proteome Discoverer software (Thermo Fisher Scientific, version 2.4.1.15) and the SequestHT search engine. Tandem mass spectra were compared with the UniProt Mus musculus database (updated November 2022) and common contaminants to identify peptides. Cysteine ​​carbamidomethylation was set as a fixed modification. For peptide identification, methionine oxidation and N-terminal acetylation of proteins were set as variable modifications. Contaminant proteins were excluded from subsequent data analysis.

[0104] 10. Western blot Western blotting was performed according to standard protocols. Protein extracts were separated by SDS-PAGE. The gel was then transferred to a nitrocellulose membrane (PDVF / filter paper interlayer, Merck Millipore, IPVH00010) for 1.5 hours. The membrane was then blocked in 5% skim milk for 1 hour and incubated overnight at 4°C with a primary antibody diluted to the appropriate concentration. The membrane was then washed three times for 15 minutes each in Tris-buffered saline containing Tween 20. Horseradish peroxidase (HRP)-labeled secondary antibody (APPLYGEN, 1:3000) was then added and incubated at room temperature for 2 hours. The following primary antibodies were used: rabbit anti-SERBP1 (Soloarbio, K109484P, 1:1000), rabbit anti-SERBP1 (Proteintech, 10729-1-AP, 1:1000), anti-GAPDH (HRP-tagged) (1:5000, Easybio, BE0034), rabbit anti-Flag (Easybio, BE2005, 1:1000), and rabbit anti-HA (Easybio, BE2008, 1:1000).

[0105] 11. Immunoprecipitation For immunoprecipitation, HEK293T cells were first cultured in 10 cm culture dishes to 70%-80% confluence, and then transfected with 5 μg of various construct plasmids using PEI. 24 hours post-transfection, HEK293T cells were treated with 10 μM MMG132 (Selleck, S2619) for 6 hours, and then lysed for 30 minutes in NP40 lysis buffer (85 mM KCl, 5 mM PIPES, 0.5% NP40) supplemented with the protease inhibitor PMSF. After centrifugation at 13,000 rpm for 15 minutes at 4°C, the supernatant (whole-cell lysis buffer) was collected. The whole-cell lysis buffer was pretreated by incubating with Protein A / G agarose beads (Santa Cruz, SC-2003) at 4°C for 1 hour. The supernatant was then incubated overnight at 4°C with the following antibodies sequentially: mouse anti-FLAG (Easybio, BE2223, 1:200), mouse anti-SERBP1 (Proteintech, 67792-1-Ig, 1:200), mouse anti-HIS (Easybio, BE2017, 1:200), and Protein A / G agarose beads for 6 hours at 4°C (mouse IgG served as a control). The precipitate was washed three times with immunoprecipitation buffer, boiled in sample buffer, and then subjected to Western blot analysis.

[0106] 12. MPTP Processing Two-month-old C57BL / 6J mice were administered BC1618 orally by gavage before, during, and after MPTP administration. The MPTP-treated group received four intraperitoneal (ip) injections of MPTP·HCl (20 mg / kg free base) dissolved in saline (Sigma-Aldrich, M0896) per day, with each injection spaced 2 hours apart.

[0107] 13. Behavioral tests Rod test A wooden instrument, 50 cm long and 1 cm in diameter, was used. The mice underwent a training exercise consisting of three tests the day before the actual test. On the day of the test, the mice were placed head-up, 3 inches from the top of the bar. The time required for them to turn and the total time to reach the bottom of the bar were recorded. The maximum time limit for testing and recording was 60 seconds.

[0108] grip test A mouse is placed on a metal grid and allowed to grasp it with its forelimbs. Once the mouse has firmly gripped the grid, its tail is pulled back until the animal releases its grip. The maximum gripping force is recorded by a force sensor before the mouse releases its grip on the grid. The peak gripping force is digitally recorded and displayed as a force value in grams (g).

[0109] Directed differentiation of midbrain dopamine neurons (mDA) Human pluripotent stem cells were digested into single cells using Accutase (Cell Technologies, AT104) and seeded at a density of 400,000 cells / cm² in Geltrex (Life Technologies, A1413201) coated culture dishes. Cells were cultured in Neurobasal medium (Life Technologies) supplemented with N2 (Stem Cell Technologies), B27 (Life Technologies), 2 mM L-glutamine, 500 ng / ml SHH C25II (R&D Systems, 464-SH), 250 nM LDN (Stemgent, 04-0074-02), 10 µM SB431542 (R&D Systems, 1614), 0.7 µM CHIR99021 (R&D Systems, 4432), and 10 µM Rock inhibitor Y-27632 (R&D Systems, 1254). This point was defined as day 0 of differentiation. Rock inhibitors were removed from day 1, and cells were cultured until day 3. From day 4, cells were exposed to different concentrations of CHIR99021 (0.7, 3, 5, and 7.5 µM) until day 10. LDN, SB431542, and SHH were removed on day 7. On day 10, the medium was replaced with Neurobasal / B27 containing L-glutamine, and BDNF (20 ng / ml; R&D Systems, 248-BD), ascorbic acid (0.2 mM; Sigma, A4034), GDNF (20 ng / ml; Peprotech, 450-10), TGF-β3 (1 ng / ml; R&D Systems, 243-B3), dibutyrylcyclic adenosine monophosphate (0.2 mM; Sigma, D0627), and CHIR99021 (3 µM) were added. On day 11, cells were digested with Accutase and reseeded at a high density (800,000 cells / cm²) in culture dishes coated with polyornithine (15 µg / ml), laminin (1 µg / ml), and fibronectin (2 µg / ml). They were then cultured in mDA differentiation medium containing L-glutamine, BDNF, ascorbic acid, GDNF, db-cAMP, and TGF-β3. From day 12, DAPT (10 µM; R&D Systems, 2634) was added to the medium, and culture continued until day 16. On day 16, cells were digested and reseeded again using the same method as day 11, and cultured in mDA differentiation medium until day 25. On day 25, cells were digested with Accutase and reseeded at a low density (200,000-300,000 cells / cm²) in mDA differentiation medium until use in subsequent experiments.

[0110] Statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). All data are expressed as mean ± standard error (mean ± SEM). Statistical significance was determined using unpaired two-tailed Student's t-test (for two groups) or one-way ANOVA followed by Tukey's multiple comparison test (for three or more groups). P < 0.05 was considered statistically significant. All representative images or photomicrographs were from experiments repeated at least three times.

[0111] Example 1: High-throughput screening of ubiquitin libraries identified BC1618 as having potent neuroprotective effects. Elevated glutamate levels have been confirmed in various central nervous system diseases, and an increasing number of studies use glutamate stimulation for drug screening (Doozandeh et al., 2016; Czapski et al., 2021; Iovino et al., 2020). To investigate the role of ubiquitination in axonal degeneration and neuronal death, we screened a library of ubiquitin compounds using an in vitro model of glutamate excitotoxicity. We screened 181 compounds by cell viability assays and found that 32 compounds significantly increased the viability of HT22 cells after glutamate exposure compared to the control group (Figure 1A). To validate these findings, we isolated primary hippocampal neurons from mice at day 0 of birth and stimulated them with 10 μM glutamate on day 10 of in vitro culture. After 10 minutes of glutamate stimulation, neurons remained viable for 3 hours under normal conditions, and then immunofluorescence staining with the neuron-specific marker NeuN and the neurite marker MAP2 was performed to assess neuronal survival and neurite integrity. Among 32 candidate compounds derived from HT22 cells, a secondary screening was conducted in primary hippocampal neurons, identifying five effective compounds that significantly protected neuronal survival and neurite integrity compared to the control group (Figures 1B-1D). Interestingly, BC1618 exhibited the strongest neuroprotective effect, and its mediated protective effect was dose-dependent. Notably, 50 μM BC1618 achieved the maximum protective effect comparable to the undamaged group, while higher concentrations showed decreased protective efficacy (Figure 1E). In summary, these data indicate that BC1618 exhibits a potent neuroprotective effect against glutamate excitotoxicity in both HT22 cells and primary neurons. Given the protective effect of BC1618 on neuronal survival and neurite integrity, we investigated whether BC1618 could promote neurite growth in the CNS. Immature hippocampal neurons were treated with DMSO or BC1618 at DIV3, and neurite growth was subsequently measured at DIV5. Notably, BC1618 significantly promoted neurite growth at relatively low doses (1 to 5 μM), but this promotion was diminished at higher doses (Figs. 1F and 1G). Specifically, 2 μM BC1618 increased the maximum length of the longest neurite to 206 µm, compared to only 95 µm in the control group (Fig. 1H). These results indicate that BC1618 enhances neurite growth in the CNS.

[0112] Example 2: BC1618 protects retinal ganglion cell survival and promotes axon regeneration after optic nerve injury. To determine whether BC1618 promotes neuronal survival and axonal regeneration in vivo, we evaluated its effects on RGC survival and optic nerve regeneration two weeks after optic nerve injury in mice. Mice were administered different concentrations of BC1618 or its solvent via intravitreal injection immediately after ONC, and were re-injected 7 days later due to decreased availability of BC1618 in the retina; the anterograde axonal tracer cholera toxin B subunit (CTB) was administered 2 days before tissue sampling (Fig. 2A). We assessed RGC survival in the retina and axonal regeneration in the optic nerve. Two weeks after injury, RGC survival was measured by counting the number of RBPMS (RNA-binding proteins with multiple splicing) positive RGCs in whole-retinal patches. We observed a significantly higher proportion of surviving RGCs in the retinas injected with 1–5 mM BC1618 compared to the DMSO group, while 20 mM BC1618 had no effect (Figs. 2B and 2C). Notably, treatment with 2 mM BC1618 increased RGC survival to 41% (Fig. 2C). Furthermore, we assessed the potential toxicity of BC1618 to RGCs, finding that at concentrations up to 20 mM, it caused a moderate 12% RGC loss (Figs. 3A and 3B). Next, we investigated the efficacy of BC1618 in optic nerve regeneration. Compared to the control group (where only a very small number of axons could cross the lesion site), BC1618 treatment (1–5 mM) significantly promoted axonal regeneration 2 weeks after ONC (Figs. 2D and 2E). Notably, 2 mM BC1618 promoted the largest axonal regeneration (>2 mm) after ONC, while 20 mM completely eliminated this regeneration effect, consistent with the concentration-dependent toxicity profile of this compound (Figs. 2D and 2E). It is noteworthy that to distinguish between regenerated and surviving axons after ONC, we used different CTB tracers before and after the injury. 48 The results showed that BC1618 not only promoted robust axonal regeneration after ONC but also significantly delayed axonal degeneration, confirming its dual therapeutic potential (Figures 4A and 4B). These findings indicate that BC1618 significantly enhances neuronal survival and axonal regeneration in damaged CNS, and its efficacy is limited to a specific dose-dependent window.

[0113] A key consideration in intraocular application of BC1618 is managing its potential inflammation-related risks. We examined whether intravitreal injection of BC1618 or the solvent would induce microglial activation or macrophage infiltration. Three days post-treatment, both damaged and undamaged retinas showed similar levels of IBA-1 positive microglia (Figure 5), indicating no significant microglial activation after ONC. Although CD68-positive macrophages increased significantly after ONC, there was no significant difference between the BC1618-treated and solvent-treated groups (Figure 5). These data clearly demonstrate that BC1618 does not induce intraocular inflammation in our study.

[0114] Example 3: BC1618 protects RGC survival and promotes axon regeneration by inhibiting FBXO48. Given the potent effects of BC1618 (an FBXO48 inhibitor) (Liu et al., 2021), we validated the core hypothesis: inhibition of Fbxo48 is both essential and sufficient for its neuroprotective function. Using intravitreal injection of AAV2 delivery, we constructed: AAV2-control, AAV2- Fbxo48-shRNA (Short hairpin RNA) and AAV2- Fbxo48 Next, we tested the knockdown. Fbxo48 Can it simulate the dual effect of BC1618 on RGC survival and axon regeneration? AAV2 virus (AAV2-control and AAV2-control) was injected intravitreally 2 weeks before the onset of ONC. Fbxo48-shRNA ), and CTB was administered 2 days prior to tissue sampling (Figure 6A). Compared with the AAV2- control group (24% RGC survival), AAV2- Fbxo48-shRNA The group retained 54% of the RGC (Figures 6B and 6D). To evaluate AAV2- Fbxo48-shRNA The long-term protective effect of treatment was assessed by detecting RGC survival at extended time points after ONC. At 1, 3, and 6 months after ONC, only 21%, 8%, and 6% of RGCs survived in the AAV-controlled group, respectively, while AAV-... Fbxo48-shRNA Treatment resulted in the preservation of 44%, 18%, and 9% of axonal regeneration, respectively (Figures 6B and 6D). Furthermore, compared to the control group, Fbxo48 knockdown significantly enhanced axonal regeneration 2 weeks after ONC, supporting the regenerative efficacy of BC1618 (Figures 6C and 6E). In summary, these results indicate that knockdown... Fbxo48Following ONC, it provides long-term protection for RGC and promotes axonal regeneration. Previous studies have shown that BC1618 interacts directly with FBXO48 without affecting its transcription or translation (Liu et al., 2021). To establish a causal epistatic relationship, we employed a gene rescue strategy: we constructed AAV2- Fbxo48 To test whether FBXO48 is an essential molecular target in antagonizing BC1618-mediated neuroprotection and axonal regeneration, AAV2- was injected two weeks prior to ONC. control or AAV2- Fbxo48 Then, mice were injected intravitreally with either BC1618 or DMSO immediately after ONC and 7 days after ONC, and injected with CTB 2 days before analysis (Fig. 7A). Notably, overexpression of Fbxo48 completely abolished BC1618-mediated post-ONC RGC protection and axonal regeneration (Figs. 7B-7E). This cross-validation confirms that inhibition of FBXO48 is a key mechanism behind the neuroprotective effect of BC1618.

[0115] Example 4: SERBP1 was identified as a downstream medium for BC1618 and FBXO48. Previous studies have confirmed that FBXO48 increases AMPK activity by stabilizing phosphorylation of AMPKα (pAMPKα) (Liu et al., 2021). To investigate whether this mechanism supports Fbxo48-mediated neuroprotection, we investigated the neuroprotective function of AICAR (a well-known AMPK activator) (Kim et al., 2016). Unexpectedly, the results showed that AICAR failed to enhance RGC survival or axonal regeneration after ONC compared with the control group, challenging the expected neuroprotective effect (Figures 8A-8E). Although the role of AMPK signaling in axonal regeneration remains controversial, our findings suggest that AMPK activation does not promote RGC survival or axonal regeneration, consistent with previous mechanistic studies (Kong et al., 2020). This unexpected finding suggests the hypothesis that FBXO48 may have other potential targets in the CNS. Subsequently, we performed proteomic analysis on BC1618-treated HT22 cells, identifying 2080 quantitative proteins (Figure 9A). Notably, the number of upregulated proteins exceeded the number of downregulated proteins, reflecting a characteristic pattern of substrate accumulation following inhibition of FBXO48 E3 ubiquitin ligase activity. We analyzed this based on fold changes, pProteins with differential abundance were ranked by fold change and p-value. The top five candidate proteins were selected based on fold change and p-value criteria. Subsequent peptide count assessment identified SERBP1 as the highest-ranking candidate protein. Western blot analysis confirmed SERBP1 as a novel target, significantly upregulated after BC1618 treatment (Figs. 9B and 9C). Consistent with in vitro findings, immunofluorescence quantification showed that in vivo delivery of BC1618 significantly upregulated SERBP1 expression in RGCs 7 days later (Figs. 9D and 9E). Furthermore, at 14 days post-injection, knockdown of Fbxo48 in RGCs via the AAV2 vector increased SERBP1 protein levels, while overexpression of Fbxo48 decreased SERBP1 protein levels (Figs. 8F and 8G). To establish the functional adequacy of SERBP1, we constructed an AAV2- vector for targeted overexpression in RGCs. Serbp1 Vector. Consistent with our hypothesis, 14 days after ONC, overexpression of SERBP1 increased RGC survival to 37%, compared to 27% in the control group (Figures 9F and 9G), while achieving axonal regeneration levels comparable to AAV2- Fbxo48-shRNA The treatment groups were comparable (Figures 9H and 9I). In summary, our data establish SERBP1 as a key downstream effector in the FBXO48 regulatory cascade. Inhibition of FBXO48 by BC1618 or gene knockdown significantly promotes neuroprotection and axonal regeneration, independent of the classical AMPK signaling pathway, by increasing SERBP1 expression.

[0116] Example 5: BC1618 and Fbxo48 knockdown support RGC axonal regeneration by increasing SERBP1 stability. To determine the necessity of SERBP1 in the FBXO48-mediated neuroprotective pathway, we administered AAV2- via intravitreal injection to BC1618-treated mice. Serbp1-shRNA Come to silence Serbp1 Expression. Virus delivery was performed 2 weeks prior to ONC, and then we evaluated the neuroprotective phenotype of BC1618 as previously described (Figure 10A). RGC survival quantification showed that, compared with AAV2- control-shRNA Compared to the BC1618 group (40%), Serbp1Knockdown only partially attenuated the BC1618-mediated protective effect (survival rate 35%) (Figures 10B and 10C). This incomplete reversal suggests that the neuroprotective effect of BC1618 on damaged RGCs involves molecular pathways beyond SERBP1. Notably, the growth-promoting effect of BC1618 was observed during knockdown. Serbp1 Then it completely disappeared. In AAV2- Serbp1-shRNA After BC1618 treatment, almost no CTB-positive regenerated axons were observed (Figure 10D), indicating that successful optic nerve regeneration requires a high level of [unclear - possibly related to optic nerve regeneration]. Serbp1 Expression. Consistent with our results, AAV2- control-shRNA In the BC1618 group, some axonal lengths reached 2 mm after ONC (Figs. 10D and 10E). Simultaneously, AAV2- was injected into the RGC two weeks prior to ONC. Serbp1-shRNA and AAV2- Fbxo48-shRNA Phenotypic results were assessed 14 days post-injury (Figure 11A). Consistent with previous data, Serbp1 The knockdown portion was weakened. Fbxo48 Knockdown-mediated RGC protection was observed, but its pro-regeneration effect was completely eliminated (Figures 11B-11E). These findings support the view that SERBP1 dysfunction completely eliminates BC1618 / FBXO48-dependent axonal regeneration, but only partially impairs RGC survival, highlighting other factors involved in RGC survival.

[0117] Example 6: FBXO48 catalyzes K63-linked ubiquitination at 52 lysine position of SERBP1. To explore the relationship between FBXO48 and SERBP1, we performed co-immunoprecipitation (Co-IP) experiments in HEK293T cells using FLAG (FBXO48-FLAG) and SERBP1 antibodies. Western blotting revealed a clear interaction between FBXO48 and SERBP1 (Fig. 12A and 12B). Based on these findings, and considering the role of FBXO48 as an E3 ligase, we investigated whether SERBP1 is a direct substrate of FBXO48. In IP experiments using HEK293T cells, compared to the control group, cells overexpressing FBXO48 showed an accumulation of ubiquitinated SERBP1 levels upon exposure to the proteasome inhibitor (MG132) (Fig. 12C). Since K48 and K63-linked ubiquitous chains are classic and typically associated with degradation, we specifically focused our analysis on these linker types. 5². Our results indicate that SERBP1 appears to bind to the K63-linked ubiquitin chain, as evidenced by the reduced SERBP1-ubiquitin signal when UB-K63R is expressed instead of UB-K48R. Figure 12 D). To determine the structural basis of this regulatory modification, we used stepwise mutagenesis. 5 ³ We located the FBXO48-mediated ubiquitination sites on SERBP1. First, through integrated analysis of ubiquitination prediction websites and public proteome datasets, we preferentially selected seven candidate lysine ubiquitination sites: K32, K52, K68, K122 (IHABP4 domain); K190, K205, K222 (HABP4 domain). Next, we attempted to locate the FBXO48 ubiquitination sites in SERBP1 using a stepwise mutagenesis strategy. We divided the seven predicted lysine residues in the SERBP1 amino acid sequence into two segments, named segments A and B. By mutating the lysine residues in each segment to arginine, we generated two SERBP1 mutants (mut A and B). We found that the four lysine residues in segment A (mut A) significantly reduced FBXO48 ubiquitination of SERBP1, while the other mutant had no such effect. By mutating lysine to arginine in segment A, we found that the mutation at lysine 52 (K52) significantly reduced FBXO48 ubiquitination of SERBP1 (Figure 12E). In summary, we determined that FBXO48 functions as an E3 ligase for SERBP1 through direct interaction and catalyzes K63-linked ubiquitination at the K52 site of SERBP1.

[0118] Example 7: BC1618 protects against cross-species PD-related neurodegeneration Given the presence of glutamate dysregulation in Parkinson's disease models (Iovino et al., 2020), we hypothesized that BC1618 exerts a disease-modifying effect in PD models by protecting substantia nigra-striatal neurons. Therefore, we evaluated whether BC1618 could protect dopamine neurons from loss in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) poisoning model (Jackson-Lewis et al., 2007). As previously reported (Park et al., 2022), we used an acute MPTP-induced male mouse model and administered BC1618 (20 mg / kg / day) for 14 days (Figure 13A). Quantitative immunofluorescence analysis showed that BC1618 treatment significantly preserved TH⁺ fibers in the striatum and TH⁺ DA neurons in the substantia nigra pars compacta (SNpc) compared to the saline group (Figures 13B-13D). Furthermore, compared to the solvent control group, BC1618 significantly improved MPTP-induced motion defects in both rod and grip tests. Figure 13 E-13F). In summary, oral administration of BC1618 protects against MPTP-induced dopaminergic neurodegeneration and behavioral deficits, highlighting the potential therapeutic efficacy of BC1618. To advance the clinical translation of BC1618, we generated midbrain dopaminergic neurons from human pluripotent stem cells to evaluate its neuroprotective effects (E-13F). Figure 13 G). Based on MPP⁺ exposure, BC1618 combination therapy significantly restored TH immune reactivity to baseline levels, while a 30% reduction was observed in the DMSO treatment group after 24 hours (G). Figure 13 (HI). Our robust in vivo mouse data and human stem cell-derived neuronal validation strongly support the clinical translation of BC1618.

[0119] Neuronal death and axonal degeneration are key pathological features of major injuries and neurological diseases (Yang et al., 2013). Neuroprotection and axonal regeneration in the central nervous system involve a multimodal process with multi-level regulation (Hilton et al., 2024; He et al., 2016). Effective neuroprotection and axonal regeneration can be triggered by activation of key molecular pathways, such as the ubiquitin-proteasome system, which directly regulates protein degradation (Schmidt et al., 2021). Here, we demonstrate that BC1618 (an FBXO48 inhibitor) is a novel therapeutic compound that prevents functional decline in acute and chronic neurological diseases. We screened a library of ubiquitin compounds in a CNS model and identified BC1618 as a neuroprotective compound that targets and inhibits FBXO48. Although the initial assessment focused on HT22 cell viability, we performed secondary validation in primary mouse hippocampal neurons, observing both neuronal survival and neurite integrity, which significantly improved the reliability of the screening. Targeting the glutamate excitotoxic pathway represents a promising frontier in future therapeutic development, with its rationale lying in its central role in neuronal pathogenesis.

[0120] Permanent disability following CNS injury stems from the loss of neuronal (terminally differentiated cells) regenerative capacity and limited regeneration of damaged axons (Mahar et al., 2018; Liu et al., 2011). Although various genetic approaches have been shown to protect RGCs and promote axonal regeneration after ONC (Park et al., 2008; Li et al., 2024; Sun et al., 2011; Wang et al., 2024), the discovery of small molecule therapies remains limited (Au et al., 2022; Au et al., 2021). Given the ability of BC1618 to protect neurons and promote neurite growth in vitro, we hypothesized that BC1618 could enhance neuronal survival and axonal regeneration in vivo. We subsequently validated in an ONC model that BC1618 achieved RGC protection and optic nerve regeneration by targeting and inhibiting FBXO48. Furthermore, BC1618 exhibited a concentration-dependent effect, with 2 mM being the optimal therapeutic concentration for intravitreal administration, while significant toxicity was observed at 20 mM. Notably, BC1618 exhibited dual therapeutic efficacy in the mouse ONC model: while promoting axonogenesis, it also alleviated damage-induced RGC axon mutations, which explains its superior regenerative efficacy compared to Fbxo48 knockdown alone.

[0121] FBXO48 belongs to the F-box protein superfamily and functions as a substrate recognition subunit in the SKP1-culin 1 F-box (SCF)E3 ligase complex. It was initially identified as a key negative regulator of pAmpkα protein levels (Liu et al., 2021). Current research on the F-box protein family in cancer is extensive, suggesting their potential as biomarkers and therapeutic targets for cancer treatment (Tekcham et al., 2020). Several F-box proteins have become important participants in the pathogenesis of CNS diseases. FBXO10 regulates the ferroptosis pathway through ubiquitination of ACSL4, thereby protecting neurons in traumatic brain injury (Bao et al., 2021). FBXO2 / FBL2, FBXO27, and FBXW11 have been reported to regulate β-amyloid protein, suggesting their involvement in Alzheimer's disease (Atkin et al., 2014; Watanabe et al., 2012; Chae et al., 2023; Sun et al., 2021). Mutations in the FBXO7 gene (T22M, R378G, and R498X) are associated with a severe autosomal recessive juvenile Parkinson's disease (PARK15) (Zhao et al., 2020; Burchell et al., 2013; Zhou et al., 2015). Furthermore, the functions of various F-box family proteins in neurons are still being elucidated. We have revealed the neuroprotective role of FBXO48 for the first time, significantly advancing our understanding of the F-box protein family as potential research and therapeutic targets in the CNS.

[0122] Given that AMPK activators failed to enhance post-ONC RGC survival or axonal regeneration, we performed proteomics screening to identify alternative targets. SERBP1 emerged as the most promising target among numerous top candidate proteins—a protein previously unreported in neuroprotection. Based on the following evidence, we demonstrate that FBXO48 functions as an E3 ligase targeting SERBP1 for ubiquitination and stabilization: First, proteomics analysis of BC1618-treated HT22 cells detected altered SERBP1 expression, with validated evidence of a significant increase in protein levels. Second, both BC1618 administration and Fbxo48 gene manipulation (knockdown or overexpression) strongly regulated SERBP1 protein levels in RGCs in vivo. Third, bidirectional immunoprecipitation experiments demonstrated a direct physical interaction between FBXO48 and SERBP1. Fourth, overexpression of FBXO48 significantly enhanced SERBP1 ubiquitination. Finally, FBXO48 specifically promotes K63-linked ubiquitination at the SERBP1 K52 site, as demonstrated by the disappearance of ubiquitination after mutations in UB-K63R and SERBP1-K52R.

[0123] In summary, the inventors have demonstrated that BC1618's inhibition of the FBXO48-SERBP1 pathway supports retinal ganglion cell survival and promotes axonal regeneration after optic nerve injury. In an MPTP-induced mouse model of Parkinson's disease (PD), administration of BC1618 significantly delayed axonal degeneration and significantly restored motor function. These experimental data support the view that BC1618 targeting the FBXO48-SERBP1 axis is a promising preventative and therapeutic strategy for CNS diseases such as ONC and PD. BC1618 could potentially serve as a pharmaceutical component for the treatment or prevention of central nervous system injuries and diseases.

[0124] Those skilled in the art to which this application pertains may make various modifications or additions to the specific embodiments described, or adopt similar methods to replace them, without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. The application of BC1618 in the preparation of central nervous system protective drugs, characterized in that, The small molecule compound BC1618 is used to prepare drugs for the prevention or treatment of central nervous system damage or neurodegenerative diseases.

2. The application according to claim 1, characterized in that, The central nervous system injury or neurodegenerative disease is selected from optic nerve injury, spinal cord injury, stroke, Alzheimer's disease, or Parkinson's disease.

3. The application according to claim 1 or 2, characterized in that, The drug exerts its neuroprotective effect by inhibiting the E3 ubiquitin ligase activity of FBXO48.

4. The application according to claim 3, characterized in that, Inhibition of the E3 ubiquitin ligase activity of FBXO48 leads to increased protein stability of its substrate SERBP1.

5. The application according to claim 4, characterized in that, The FBXO48 catalyzes the K63-linked ubiquitination of lysine (K52) at position 52 of the SERBP1 protein.

6. The application according to any one of claims 1 to 5, characterized in that, The preventive or therapeutic effects of the drug include at least one of the following: (a) improving the survival rate of damaged neurons; (b) promoting the regeneration of damaged neuronal axons; (c) delaying the degeneration of damaged neuronal axons; (d) maintaining or restoring neural circuit function; and (e) improving motor dysfunction.

7. The application according to claim 6, characterized in that, The drug is used to treat optic nerve injury, and its effects include increasing the survival rate of retinal ganglion cells (RGCs) and promoting their axonal regeneration.

8. The application according to claim 6, characterized in that, The drug is used to treat Parkinson's disease, and its effects include protecting the survival of dopaminergic neurons in the substantia nigra pars compacta, preventing the loss of striatal dopaminergic fibers, and improving motor behavior deficits.

9. A pharmaceutical composition comprising a therapeutically effective amount of a small molecule compound BC1618, a pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate or crystal form thereof, and one or more pharmaceutically acceptable carriers or excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, Its dosage forms include tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections, inhalants, or sprays.

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