Use of lassbio-1135 as a mitochondrial autophagy agonist in neurodegenerative diseases

Abstract

The application belongs to the technical field of biological medicine, and discloses application of LASSBio-1135 as a mitochondrial autophagy agonist in neurodegenerative diseases. The application discloses a new medical use of LASSBio-1135, and finds that LASSBio-1135 can activate the AMPK / ULK1 pathway to promote mitochondrial autophagy and selectively remove damaged or dysfunctional mitochondria. The application shows through experiments that LASSBio-1135 can significantly reduce the disease phenotype of CK12 nematode models. It is indicated that LASSBio-1135 can be applied to preparation of drugs for treating various neurodegenerative diseases related to mitochondrial dysfunction, and is especially suitable for treatment of Alzheimer's disease, and is expected to be developed into a new type of drug for promoting mitochondrial autophagy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of LASSBio-1135 as a mitochondrial autophagy agonist in neurodegenerative diseases. Background Technology

[0002] Mitophagy is a specific autophagic process in which autolysosomes selectively clear damaged or abnormal mitochondria. It is primarily regulated by key pathways such as PINK1 / Parkin. This process is crucial for maintaining cellular energy homeostasis, alleviating oxidative stress, and inhibiting apoptosis, especially in high-energy-consuming neurons, where it is a core mechanism for mitochondrial quality control. The core pathological feature of neurodegenerative diseases (such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD)) is the progressive death of specific neuronal groups. Their pathogenesis is closely related to mitochondrial dysfunction, and defective mitophagy is a key driver of the development and progression of these diseases. In pathological conditions, abnormal PINK1 / Parkin pathway function or impaired autophagosome formation prevents the timely clearance of damaged mitochondria, leading to insufficient ATP production, excessive accumulation of reactive oxygen species (ROS), and aggregation of neurotoxic proteins, forming a vicious cycle of "energy crisis-oxidative stress." This ultimately exacerbates synaptic dysfunction and neuronal apoptosis, driving disease progression. Current treatments for neurodegenerative diseases still have limitations. Targeting the mitophagy pathway to repair mitochondrial quality control function has become an important direction for developing treatment options for neurodegenerative diseases, with clear clinical needs and application value.

[0003] LASSBio-1135 is an imidazo(1,2-a)pyridine derivative designed and synthesized by the LASSBio laboratory in Brazil using a molecular hybridization strategy. As a multi-target bioactive molecule, its core mechanism of action includes non-competitive antagonism of the TRPV1 channel (IC50). 50 Approximately 580 nM), inhibiting TNF-α release (IC50). 50 Approximately 546 nM) and inhibition of COX-2 activity (IC50). 50=18.5 μM), and can exert a synergistic regulatory effect by downregulating p38 MAPK phosphorylation. LASSBio-1135 has clear in vitro and in vivo pharmacological activities. Oral administration can significantly reverse capsaicin-induced thermal hyperalgesia, reduce carrageenan-induced inflammatory edema, and reduce mechanodysphagia in neuropathic pain models, without the high fever side effect common in traditional TRPV1 antagonists. It also shows potential roles in the regulation of the inflammatory microenvironment and neuroprotective pathways, providing an experimental basis for its application in neurodegenerative diseases and inflammation-related conditions. It possesses advantages in drug development due to its well-defined structure, clear target, convenient administration, and good safety profile. Currently, there are no clear reports on the application of LASSBio-1135 in drug preparation for neurodegenerative diseases by activating the AMPK / ULK1 pathway to promote mitophagy. Summary of the Invention

[0004] The first aspect of the present invention aims to provide the use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of a treatment for the prevention and / or treatment of neurodegenerative diseases.

[0005] A second aspect of the present invention aims to provide the use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of a mitophagy agonist.

[0006] A third aspect of the present invention is to provide a drug.

[0007] The fourth aspect of this invention aims to provide a method for promoting mitochondrial autophagy in vitro.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of a preventive and / or therapeutic agent for neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, or frontotemporal dementia.

[0009] In some embodiments of the present invention, the LASSBio-1135 or a pharmaceutically acceptable derivative thereof promotes mitophagy by activating the AMPK / ULK1 pathway to prevent and / or treat neurodegenerative diseases.

[0010] In some embodiments of the present invention, the pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and cocrystals of LASSBio-1135.

[0011] In some embodiments of the present invention, the pharmaceutical salt of LASSBio-1135 includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0012] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0013] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0014] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0015] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0016] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0017] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0018] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0019] In some embodiments of the present invention, the salt formed with the acidic amino acid includes at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0020] In some embodiments of the present invention, the effective dose of LASSBio-1135 or a pharmaceutically acceptable derivative thereof is 5-50 μM, such as any value or a range of any two of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μM.

[0021] A second aspect of the invention provides the use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of a mitophagy agonist.

[0022] In some embodiments of the present invention, the effective dose of LASSBio-1135 or a pharmaceutically acceptable derivative thereof is 5-50 μM, such as any value or a range of any two of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μM.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and cocrystals of LASSBio-1135.

[0024] In some embodiments of the present invention, the pharmaceutical salt of LASSBio-1135 includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0025] A third aspect of the present invention provides a medicament comprising LASSBio-1135 or a pharmaceutically acceptable derivative thereof and pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, binders, disintegrants, lubricants, flavoring agents, colorants, flavor masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers.

[0027] In some embodiments of the present invention, for ease of administration, LASSBio-1135 or its pharmaceutically acceptable derivatives may be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic regulators (such as sodium chloride and glucose).

[0028] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0029] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0030] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0031] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0032] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0033] In some embodiments of the present invention, the drug also includes combination drugs.

[0034] In some embodiments of the present invention, the combination drugs include drugs for treating neurodegenerative diseases, such as lencanemab, donepezil, rivastigmine, galantamine, risperidone, olanzapine, quetiapine, etc. for treating Alzheimer's disease, and levodopa, carbidopa, pramipexole, ropinirole, selegiline, rasagiline, entacapone, amantadine, trihexyphenidyl, etc. for treating Parkinson's disease.

[0035] A fourth aspect of the present invention provides a method for promoting cellular mitophagy in vitro, the method comprising the step of treating cells with LASSBio-1135 or a pharmaceutically acceptable derivative thereof.

[0036] In some embodiments of the invention, the treatment includes culturing cells using a culture medium containing LASSBio-1135 or a pharmaceutically acceptable derivative thereof.

[0037] The beneficial effects of this invention are: This invention discloses a novel pharmaceutical use of LASSBio-1135, revealing that it can activate the AMPK / ULK1 pathway to promote mitophagy and selectively clear damaged or dysfunctional mitochondria. Experiments demonstrate that LASSBio-1135 significantly alleviates the disease phenotype in a CK12 nematode model. This suggests that LASSBio-1135 could be used to develop drugs for treating various neurodegenerative diseases related to mitochondrial dysfunction, particularly Alzheimer's disease, and holds promise for development as a novel drug promoting mitophagy. Attached Figure Description

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 LASSBio-1135 can promote mitophagy; A shows the flow cytometry results of TMRE in HeLa cells after drug treatment (concentration: μM, treatment time: 24 h), with CCCP as a positive control; B shows the immunofluorescence results of mt-Keima-YFP-Parkin HeLa cells after drug treatment (treatment time: 24 h), with CCCP as a positive control (5 μM, 2 h), scale bar: 50 μm; C shows the flow cytometry results of mt-Keima-YFP-Parkin HeLa cells after drug treatment, with the same treatment time and concentration as B; D shows the Western blot results of protein extraction from HeLa cells after drug treatment, with the same treatment time and concentration as B. (Figure...) express p <0.05, express p<0.01.

[0039] Figure 2 LASSBio-1135 regulates mitophagy and autophagy through the AMPK / ULK1 signaling pathway. In Figure A, HEK293 cells with WT and AMPK α1 / α2 double knockout (AMPK DKO) were treated with the drug (10 μM, 24 h) and Western blot analysis of AMPK / ULK1 pathway-related proteins. Figure B shows mitochondrial membrane proteins detected by Western blot analysis in WT and AMPK DKO cells. Figure C shows autophagy-related proteins detected by Western blot analysis in WT and AMPK DKO cells. express p <0.05, express p <0.01.

[0040] Figure 3 The effect of LASSBio-1135 on the lifespan of nematodes with Alzheimer's disease.

[0041] Figure 4 The effect of LASSBio-1135 on the memory ability of Alzheimer's disease nematodes. In the figure, ns indicates no significant difference. express p <0.0001. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0044] The molecular formula of LASSBio-1135 is C 18 H 14 N4, the structural formula is: The CAS number is 852453-71-5.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1: Effects of LASSBio-1135 on mitophagy HeLa or mt-Keima cells were seeded at an appropriate density in culture plates (1 × 10⁶ cells per well for a six-well plate). 5 Cells / well, 96-well plate at 1×10 4 Cells were seeded in culture medium (10% FBS, 90% DMEM) and cultured overnight at 37°C with 5% CO2 to allow them to adhere. The next day, LASSBio-1135 (denoted as LASS) was added to a specified concentration for treatment (in this experiment, LASS at final concentrations of 5, 10, and 15 μM and an equal volume of DMSO served as blank controls, and CCCP (carbonyl cyanide m-chlorophenylhydrazone) at a final concentration of 5 μM served as a positive control). After 24 h of treatment, the drug-containing culture medium was discarded, and the cells were gently washed once with pre-warmed PBS. The next step of the experiment was performed according to either the AD method.

[0047] A: After drug treatment, cells were digested with trypsin-EDTA. After cell detachment, serum-containing medium was added to stop digestion. The cells were gently pipetted and transferred to centrifuge tubes, and the supernatant was discarded after centrifugation. The collected cells were resuspended in fresh, serum-free DMEM, and tetramethylrhodamine ethyl ester (TMRE) was added to a final concentration of 100 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 30 min in the dark. After staining, the cells were collected by centrifugation, washed twice with pre-warmed PBS to remove unbound free probes, and finally resuspended in PBS buffer containing 1% FBS. The cells were then filtered through a 40 μm filter into flow cytometry tubes. An unstained control group (used for gating) and a positive control group were also set up. Detection was performed using the PE channel of the flow cytometer (excitation wavelength 543 nm, emission wavelength approximately 570-590 nm), with 10,000 cells collected per tube. During data analysis, single-cell populations were first identified, and debris and adherent cells were excluded. Then, the unstained group was used as a control to delineate TMRE-positive cell phyla. The mean fluorescence intensity (MFI) of cells in each treatment group was calculated as a relative quantitative indicator reflecting mitochondrial membrane potential.

[0048] B: After drug treatment, high-content live-cell imaging analysis was performed: The cell plate was placed on a high-content microscope equipped with a live-cell culture device (37℃, 5% CO2), and images were acquired using a 40× objective lens. mKeima cells in a neutral pH environment (emission collection range 550-600 nm, appearing green) were excited by a 405 nm laser, and mKeima cells in an acidic lysosomal environment (emission collection range 570-650 nm, appearing red) were excited by a 561 nm laser. At least 20 fields of view were randomly acquired from each well. The red / green fluorescence intensity ratio of each cell was calculated using high-content image analysis software (Harmony) as a quantitative indicator of autophagy / mitochondrial autophagy activity.

[0049] C: For flow cytometry analysis, after drug treatment, the culture medium was discarded, and the cells were gently washed once with pre-warmed PBS. After trypsin digestion and neutralization with serum-containing culture medium, the cells were collected by centrifugation, resuspended in PBS buffer containing 1% FBS, filtered through a 40 µm filter membrane into flow cytometry tubes, and loaded onto a BD FACSCelesta flow cytometer with excitation at 405 nm and 561 nm. The emission signals were collected in the 610 / 20 nm channels, with 10,000 cells collected per tube. For data analysis, single-cell populations were first circled using FSC-A / SSC-A, and then mKeima positive gates were set to exclude negative cells. The green fluorescence intensity under 405 nm excitation represented the neutral environment (cytoplasm / normal mitochondria), and the red fluorescence intensity under 561 nm excitation represented the acidic environment (autophagosomes / lysosomes). The red / green fluorescence intensity ratio was calculated to reflect autophagy / mitochondrial autophagy flux.

[0050] D: After drug treatment, discard the culture medium, wash cells twice with pre-cooled PBS, and add 100 μL of RIPA lysis buffer (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA) containing 1× protease inhibitor and 1× phosphatase inhibitor (i.e., Protease and Phosphatase Inhibitor Cocktail, purchased from MCE, catalog number HY-K0013) to each well of a six-well plate. Lyse on ice for 30 min, then centrifuge at 12,000 rpm and 4℃ for 15 min to collect the supernatant. Determine protein concentration using a BCA protein quantification kit, add an equal volume of protein to 5× SDS loading buffer, and boil for 10 min to denature the protein. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane using a wet transfer method under constant current conditions of 250 mA for 120 min. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. Then, anti-MFN2 antibody (Mitofusin-2, purchased from CST, 9482S) (dilution 1:1000) and anti-TIM23 antibody (TIM23 Polyclonal antibody, purchased from Proteintech, 11123-1-AP) (dilution 1:1000) were added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 min each time, and then incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:5000) at room temperature for 1 h. The membrane was then washed three more times with TBST. Finally, enhanced chemiluminescence (ECL) substrate development was used, and chemiluminescence signals were acquired using a gel imaging system. α-Tubulin was used as an internal reference protein for band grayscale analysis to calculate the relative expression levels of MFN2 and TIM23.

[0051] The results are as follows Figure 1 As shown, the small molecule compound LASSBio-1135 can promote mitophagy in HeLa cells and mt-Keima cells without affecting mitochondrial membrane potential.

[0052] Example 2: LASSBio-1135 regulates mitophagy and autophagy through the AMPK / ULK1 signaling pathway HEK293 cell lines and AMPK double knockout (DKO) HEK293 cells were treated with the small molecule compound LASSBio-1135 (these cells were donated by Professor Shen Hanming's research group at our university; they were constructed using a Cas9 nickase editing strategy, with paired double-stranded gRNAs targeting exon 1 of the human PRKAA1 and PRKAA2 genes designed and cloned into the pX462 vector; transfection of the corresponding gRNA vector alone could obtain AMPK α1 or α2 single knockout cells, and co-transfection of the two vectors could construct AMPK α1 / α2 double knockout cells; after 48 h of transfection, the cells were screened for 2 days with puromycin to obtain a knockout cell pool, and single-cell cloning was continued; after immunoblotting, monoclonal DKO cell lines with complete AMPK knockout were obtained). The study investigated whether LASSBio-1135 activated AMPK and regulated the AMPK / ULK1 signaling pathway.

[0053] The experimental procedure is as follows: HEK293 cells from WT and AMPK DKO were cultured at a rate of 1×10⁻⁶. 5Cells were seeded in six-well plates and cultured overnight at 37°C and 5% CO2 to allow them to adhere. The next day, cells were treated with a final concentration of LASSBio-1135 (denoted as LASS) (in this experiment, final concentrations of 5 μM LASS, 10 μM LASS, and an equal volume of DMSO served as blank controls) in medium (10% FBS, 90% DMEM). After 24 h of treatment, the drug-containing medium was discarded, and the cells were gently washed once with pre-warmed PBS. Add an appropriate amount of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (100 μL per well of RIPA lysis buffer containing 1× protease inhibitor and 1× phosphatase inhibitor (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA), lyse on ice for 30 min, then centrifuge at 12,000 rpm, 4℃ for 15 min and collect the supernatant. Determine protein concentration using a BCA protein quantification kit. Take an equal volume of protein and add 5× SDS loading buffer, boil for 10 min to denature the protein. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transfer the protein samples to a PVDF membrane using wet transfer at a constant current of 250 mA for 120 min. Block with 5% skim milk powder at room temperature for 1 h, then add anti-MFN2 antibody (dilution ratio 1:1000, CST, ...). Anti-TIM23 antibody (dilution 1:1000, Proteintech, 11123-1-AP), anti-p-AMPK antibody (dilution 1:1000, CST, 2535T), anti-AMPK antibody (dilution 1:1000, CST, 2532S), anti-p-ULK1 antibody (dilution 1:1000, CST, 5869T), anti-ULK1 antibody (dilution 1:1000, CST, 8054T), anti-LC3 antibody (dilution 1:1000, CST, 12513S), and anti-SQSTM1 / p62 antibody (dilution 1:1000, abcam, AB109012) were incubated overnight at 4°C. The next day, the membrane was washed three times with TBST, 10 μL each time. Add horseradish peroxidase-labeled secondary antibody (dilution ratio 1:5000) and incubate at room temperature for 1 h. Wash the membrane three times with TBST. Finally, develop the membrane using enhanced chemiluminescence (ECL) substrate and acquire chemiluminescence signals using a gel imaging system. Use α-Tubulin as an internal control protein for band grayscale analysis to calculate the relative expression levels of MFN2, TIM2, p-AMPK, AMPK, p-ULK1, ULK1, LC3, and p61. Calculate the standardized p-AMPK / AMPK and p-ULK1 / ULK1 levels.

[0054] Experimental results are as follows Figure 2 As shown in Figure A, the small molecule compound LASSBio-1135 can activate AMPK and regulate the AMPK / ULK1 signaling pathway in both HEK293 cell lines and AMPK double knockout (DKO) HEK293 cells.

[0055] Example 3 HEK293 cell lines and AMPK double knockout (DKO) HEK293 cells were treated with the small molecule compound LASSBio-1135 to investigate whether LASSBio-1135 regulates mitophagy and autophagy through the AMPK / ULK1 signaling pathway. The specific experimental procedure was the same as in Example 2.

[0056] The results showed that in HEK293 cells, the protein expression of mitochondrial autophagy-related proteins (mitochondrial outer membrane protein MFN2 and mitochondrial inner membrane protein TIM23) and autophagy-related proteins (p62 and LC3) was significantly different after treatment with the small molecule compound LASSBio-1135 compared with cells that were not treated with the drug. Figure 2 In AMPK DKO HEK293 cells, after treatment with the small molecule compound LASSBio-1135, the protein expression of mitochondrial outer membrane protein MFN2, mitochondrial inner membrane protein TIM23, and autophagy-related proteins p62 and LC3 were not significantly different from those in untreated AMPK DKO HEK293 cells. Figure 2 (Middle B and C). This suggests that LASSBio-1135 regulates mitophagy and autophagy through the AMPK / ULK1 signaling pathway.

[0057] Example 4: Construction of the CK12 transgenic nematode model A hermaphroditic Caenorhabditis elegans AD disease model (i.e., the CK12 transgenic nematode model) was constructed using molecular cloning technology. The specific experimental procedure is as follows: First, the amplification and preparation of the target gene fragments were carried out. Based on the corresponding template plasmid, specific amplification primers were designed to amplify three core gene fragments, including the paex-3 promoter fragment for driver-specific expression, the human mutant Tau protein coding sequence hTau-4R1N (P301L), and the pmyo-2::GFP fluorescently labeled fragment as a positive selection marker. After amplification, the purity and size of the amplification products were detected by agarose gel electrophoresis, and the target fragments were purified and recovered using a gel extraction kit to remove non-specific amplification fragments, primer dimers, and impurities, ensuring the template purity for subsequent cloning experiments.

[0058] Molecular cloning of the recombinant expression plasmid was then performed. The purified paex-3 promoter fragment, hTau-4R1N(P301L) target gene fragment, pmyo-2::GFP marker fragment, and empty plasmid vector were subjected to double digestion with matched restriction endonucleases. Incubation was strictly performed according to the enzyme digestion system ratio and temperature / time conditions to induce complementary sticky ends between the vector and each target fragment. After digestion, the digestion products were purified again to remove enzyme proteins and buffer impurities. The treated vector backbone and the three target fragments were mixed in appropriate molar ratios, and T4 DNA ligase and its matching ligation buffer were added. The mixture was incubated overnight at a constant temperature to ensure precise splicing of each gene fragment with the vector, resulting in a complete recombinant expression plasmid. The ligation product was transformed into competent *E. coli*, plated on corresponding antibiotic-resistant solid medium, and incubated overnight at a constant temperature. Single colonies were picked for PCR identification and plasmid sequencing verification. Positive recombinant plasmids with completely correct sequences and no mutations were screened. After large-scale amplification, high-purity plasmids were extracted for subsequent nematode microinjection experiments.

[0059] After plasmid construction, microinjection and transgenic line screening and purification of *C. elegans* were carried out. Wild-type N2 hermaphroditic *C. elegans* nematodes in good growth condition and at the young adult stage were selected as recipient materials. The validated recombinant plasmid was prepared into an injection system of appropriate concentration and precisely introduced into the gonadal cells of N2 nematodes using microinjection technology. After injection, the nematodes were transferred to normal nematode culture medium for isothermal culture. After oviposition and progeny hatching, screening was performed using stereofluorescence microscopy. Positive transgenic progeny nematodes with green fluorescent signals were selected based on the expression characteristics of green fluorescent protein (GFP) driven by the pmyo-2 promoter, while wild-type individuals without fluorescence were discarded.

[0060] Finally, the transgenic nematode strain was stabilized, purified, and the model was finalized. Positive fluorescent nematodes obtained from the initial screening were individually selected and cultured. Through multiple generations of continuous hybridization, passage purification, and fluorescence screening, heterozygous individuals and nematodes with unstable expression were continuously eliminated. Ultimately, a homozygous hermaphroditic transgenic nematode strain with stable genetic traits, capable of stably inheriting the hTau-4R1N(P301L) mutant gene, and carrying the GFP fluorescent marker was obtained. This strain is the target CK12 transgenic *C. elegans* AD disease model (i.e., the CK12 transgenic nematode model), which can be used for subsequent experimental studies on the mechanisms and functions related to Tau protein pathogenesis.

[0061] Example 5: Effects of LASSBio-1135 on the lifespan of diseased nematodes The effect of LASSBio-1135 on the lifespan of diseased nematodes was investigated using LASSBio-1135 treatment. The specific experimental procedure is as follows: Select healthy, unmated CK12 nematodes (i.e., the Tau protein-induced nematode model strain constructed in Example 4) or L4 stage hermaphroditic young adults of N2 nematodes with consistent growth status, and place them in a solution of Escherichia coli OP50 (OD200). 600 On NGM medium (containing 3 g sodium chloride, 17 g agar, 2.5 g peptone and 975 mL water, sterilized in an autoclave, then 1 mL of 1M calcium chloride solution, 1 mL of 1M magnesium sulfate solution, 1 mL of 5 mg / mL cholesterol and 25 mL of 1 M potassium phosphate) plates containing OP50 bacterial suspension in the 0.8-1.2 range, 40 μL / plate for 35 mm plates, 100 μL / plate for 6 mm plates, and 400 μL / plate for 100 mm plates, were pre-cultured at 20°C for 12 h to induce oviposition. All oviposition adults were then transferred to fresh OP50 plates and incubated at 20°C in the dark for 2 h. After the incubation period, all adults were removed from the plates using a picking needle and transferred to discarded plates, leaving only the freshly laid eggs on the plates. The plates were then cultured for another 42 h until the larvae reached the L4 instar. M9 buffer (containing 5 g sodium chloride, 3 g potassium dihydrogen phosphate, 4.6 g sodium chloride, 17 g agar, 2.5 g peptone and 975 mL water, sterilized in an autoclave, then 1 mL of 1M calcium chloride solution, 1 mL of 1M magnesium sulfate solution, 1 mL of 5 mg / mL cholesterol and 25 mL of 1 M potassium phosphate) was used. (Prepared by sterilizing disodium hydrogen phosphate, 1 mL of 1 M magnesium sulfate solution, and water to a final volume of 1 L, and then using the solution for later use) The nematodes were rinsed, collected by centrifugation, and residual culture medium was removed to obtain synchronized nematode larvae. The synchronized N2 and CK12 nematodes were randomly divided into two groups, with three biological replicates in each group: ① N2 and CK12 control group: N2 and CK12 nematodes were inoculated onto NGM agar plates containing E. coli OP50, with an equal volume of DMSO (consistent with the solvent concentration in the drug group); ② 40 μmol / L LASSBio-1135 treatment group: N2 and CK12 nematodes were inoculated onto NGM agar plates containing E. coli OP50 and 40 μmol / L LASSBio-1135. Both groups of nematodes were incubated at 20℃ for 48 h, with adequate food provided during this period. To observe the mortality of nematodes, gently touch the head of the nematode with a nematode hook and observe its state under a stereomicroscope. Nematodes that do not swallow are considered dead.

[0062] The results are as follows Figure 3 As shown, the small molecule compound LASSBio-1135 can significantly prolong the lifespan of CK12 transgenic nematodes at 40 μM.

[0063] Example 6: Effects of LASSBio-1135 on the memory ability of diseased nematodes The effect of LASSBio-1135 on the memory ability of diseased nematodes was investigated using LASSBio-1135 treatment. The specific experimental procedure is as follows: Healthy, unmated CK12 nematodes (i.e., the Tau protein-induced nematode model strain constructed in Example 4) or hermaphroditic young L4 stage N2 nematodes with consistent growth were selected and placed on NGM medium (3 g sodium chloride, 17 g agar, 2.5 g peptone and 975 mL water were weighed, and after sterilization in an autoclave, 1 mL of 1 M calcium chloride solution, 1 mL of 1 M magnesium sulfate solution, 1 mL of 5 mg / mL cholesterol and 25 mL of 1 M potassium phosphate were added) plated with Escherichia coli OP50. The plates were pre-cultured at 20°C for 12 h to allow them to mature and become oviparous. All oviparous adults were then transferred to a brand new OP50 plate and incubated at 20°C in the dark for 2 h. After the incubation period, all adults on the plate were picked off with a needle and transferred to a discarded plate, leaving only the freshly laid eggs on the plate. The plates were then cultured for another 42 h until they reached the L4 instar larval stage. The nematodes were washed with M9 buffer, centrifuged, and the residual culture medium was removed to obtain synchronized nematode larvae. Synchronized N2 and CK12 nematodes were randomly divided into two groups, with three biological replicates in each group, for chemotactic memory training.

[0064] Chemotactic memory training: ① Naïve group: After culture, the two groups of nematodes (N2 and CK12) were rinsed with M9 buffer and transferred to NGM medium plates without E. coli OP50 and starved for 2 h; ② Drug group: After culture, the two groups of nematodes (N2 and CK12) were rinsed with M9 buffer and transferred to NGM medium plates without E. coli OP50. 10 μL of isoamyl alcohol (IA) was added to the inner surface of the lid and gently spread. The nematodes were starved for 2 h to establish a conditioned memory association of "IA odor presence → no food".

[0065] Chemotaxis memory assay: ① Plate preparation: Take a sterile 9 cm culture dish, pour in NGM medium cooled to about 50℃, and after solidification, add 10 μL of isoamyl alcohol (chemokine source) and 5 μL of M9 buffer (blank control) to opposite ends of the plate, respectively. Let stand at room temperature for 30 min to allow the solution to diffuse and form a concentration gradient; ② Nematode inoculation: Use a pipette to pick up about 200 trained nematodes and add them to the center of the assay plate. Let stand at room temperature in the dark for 2 h; ③ Chemotaxis index statistics: Use a stereomicroscope to count the number of nematodes at the IA drop site (chemotactic zone), the M9 buffer drop site (blank area), and the center of the plate. Calculate the chemotaxis index (CI) using the following formula: Chemotaxis index (CI) = (Number of nematodes in chemotactic zone - Number of nematodes in blank area) / Number of nematodes in the plate. The closer the CI value is to 1, the stronger the chemotaxis of the nematodes to sodium chloride and the better the memory retention effect.

[0066] The results are as follows Figure 4As shown, without drug intervention, CK12 memory was worse than N2. After drug treatment, the memory ability of CK12 nematodes was significantly improved compared with that of CK12 nematodes without drug treatment.

[0067] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of a treatment for the prevention and / or treatment of neurodegenerative diseases, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, or frontotemporal dementia.

2. The application according to claim 1, characterized in that, The LASSBio-1135 or its pharmaceutically acceptable derivatives promote mitophagy by activating the AMPK / ULK1 pathway to prevent and / or treat neurodegenerative diseases.

3. The application according to claim 1 or 2, characterized in that, The pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and cocrystals of LASSBio-1135.

4. The application according to claim 3, characterized in that, The medicinal salt includes at least one of the following: metal salt, ammonium salt, salt formed with inorganic acid, salt formed with organic base, salt formed with organic acid, salt formed with basic amino acid, and salt formed with acidic amino acid.

5. The application according to claim 4, characterized in that, The metal salt includes alkali metal salts and alkaline earth metal salts; and / or, the alkali metal salt includes at least one of sodium salts and potassium salts; And / or, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid; And / or, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; And / or, the salt formed with the basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, ornithine; And / or, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

6. The application according to claim 4 or 5, characterized in that, The effective dose of LASSBio-1135 or its pharmaceutically acceptable derivative is 5-50 μM.

7. The use of LASSBio-1135 or a pharmaceutically acceptable derivative thereof in the preparation of mitochondrial autophagy agonists.

8. The application according to claim 7, characterized in that, The effective dose of LASSBio-1135 or its pharmaceutically acceptable derivative is 5-50 μM.

9. A drug, characterized in that, The drug includes LASSBio-1135 or a pharmaceutically acceptable derivative thereof and pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of the following: fillers, binders, disintegrants, lubricants, flavoring agents, colorants, flavor masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers. Preferably, the drug further includes combination drugs; Preferably, the combination drugs include drugs used to treat neurodegenerative diseases.

10. A method for promoting mitochondrial autophagy in vitro, characterized in that, The method includes the step of treating cells with LASSBio-1135 or a pharmaceutically acceptable derivative thereof.

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