Use of chlorogenic acid in the preparation of a drug for treating neuronal intranuclear inclusion disease

Abstract

This invention provides the application of chlorogenic acid in the preparation of drugs for treating intranuclear inclusion body disease (NIID), belonging to the field of pharmaceutical technology. This invention utilizes widely available sources with abundant safety data. Compared to novel chemical entities, chlorogenic acid, with its lower development risk, is used as a raw material. It has been found that chlorogenic acid can enhance the interaction between polyG protein and the E3 ubiquitin ligase TRIM21, forming a stable TRIM21-chlorogenic acid-polyG ternary complex. This promotes polyG ubiquitination and proteasome-dependent degradation, thereby reducing intracellular inclusion body burden and alleviating NIID from its pathological root cause. This provides a safe, effective, and highly specific new targeted therapeutic strategy for NIID.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of chlorogenic acid in the preparation of drugs for treating neuronal intranuclear inclusion body disease. Background Technology

[0002] NOTCH2NLC Neuronal intranuclear inclusion disease (NIID) is a condition caused by... NOTCH2NLC Neurodegenerative diseases caused by abnormal amplification of the GGC gene duplication are characterized by the aggregation of abnormal polyglycine (polyG) proteins, which form inclusion bodies in or around the cell nucleus.

[0003] Currently, clinical treatment for NIID is mainly limited to symptomatic and supportive care, with no disease-modifying drugs available to improve disease progression. Existing treatments cannot improve or reverse neurological damage caused by the pathological deposition of polyG inclusion bodies, leading to poor patient prognosis. Targeted degradation of pathogenic inclusion bodies is a pressing technical challenge in this field, and to date, no small molecule drugs based on molecular glue mechanisms for targeted degradation of polyG inclusion bodies have been approved for clinical use. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of chlorogenic acid in the preparation of drugs for treating intranuclear inclusion body disease of neurons. This invention discovers that chlorogenic acid can act as a molecular glue to target and promote the ubiquitination and degradation of polyG inclusion bodies in NIID by the E3 ubiquitin ligase TRIM21, thereby alleviating NIID from its pathological root cause and providing a safe, effective, and highly specific new targeted therapeutic strategy for NIID.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of chlorogenic acid in the preparation of drugs for treating neuronal intranuclear inclusion body disease.

[0006] Preferably, the concentration of chlorogenic acid is 0.4~50 μM.

[0007] Preferably, the neuronal intranuclear inclusion body disease is caused by NOTCH2NLC This is caused by abnormal amplification of the GGC gene duplication.

[0008] Preferably, the dosage form of the drug is an oral formulation.

[0009] Preferably, the chlorogenic acid is any one of chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid.

[0010] The present invention also provides a medicament for treating neuronal intranuclear inclusion body disease, the medicament being chlorogenic acid and its pharmaceutically acceptable carrier.

[0011] Preferably, the chlorogenic acid is any one of chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid.

[0012] Preferably, the concentration of chlorogenic acid is 0.4~50 μM.

[0013] Compared with existing technologies, this invention has the following beneficial effects: This invention is the first to utilize a molecular glue mechanism to promote polyG degradation. This invention uses widely available sources with abundant safety data. Compared to novel chemical entities, it utilizes chlorogenic acid, a lower-risk raw material. It has been found that chlorogenic acid can enhance the interaction between polyG protein and the E3 ubiquitin ligase TRIM21, forming a stable TRIM21-chlorogenic acid-polyG ternary complex, promoting polyG ubiquitination and proteasome-dependent degradation, thereby reducing intracellular inclusion body load. This can alleviate NIID from its pathological root cause, providing a safe, effective, and highly specific new targeted therapy strategy for NIID. Attached Figure Description

[0014] Figure 1 For computer-based virtual screening and calculation of binding energy of the TRIM21-CA-polyG ternary complex; Figure 2 To verify the target binding of CA to TRIM21 using cell thermal shift assays, (a) is a representative Western blot image of the cell thermal shift assay, and (b) is an image of the target binding of CA to TRIM21. Figure 2 In (a), the gray values ​​of the TRIM21 protein band were used for quantification, and the relative melting temperature curve was plotted after normalization with GAPDH. Figure 3 To detect interaction affinity in surface plasmon resonance experiments; Figure 4 The effect of 10 μM chlorogenic acid intervention on polyG inclusion body deposition in HEK293 cells, where (a) shows HEK293 cells transfected with polyG. 17 -GFP or polyG 92 (a) Representative image of immunofluorescence staining after GFP, scale bar = 50 μm, scale bar = 20 μm in magnified image; (b) HEK293 cells transfected with polyG 17 -GFP or polyG 92 - Representation of Western blot after GFP transfection; (c) shows HEK293 cells transfected with polyG. 92 Representative Western blot images of patients treated with GFP followed by 10 μM chlorogenic acid; (d) shows... Figure 4 (c) Quantitative analysis of polyG aggregates; (e) for Figure 4 (c) Quantitative analysis of polyG monomers; (f) Western blot representation of chlorogenic acid effect on polyG after MG132 intervention; (g) PolyG transfection of HEK293 cells. 92 Representative immunofluorescence staining image after GFP treatment with 10 μM chlorogenic acid, scale bar = 100 μm; (h) represents... Figure 4 Statistical analysis of the ratio of aggregate number to cell nucleus number in (g) of the sample; express p <0.05, express p <0.01; Figure 5 The effect of 10 μM chlorogenic acid intervention on polyG inclusion body deposition in dorsal forebrain organoids is shown in the following images: (a) representative bright-field images of hiPSC-derived dorsal forebrain organoids from NIID patients at different culture stages; (b) representative immunofluorescence staining images of mature dorsal forebrain organoids after 7 days of 10 μM chlorogenic acid intervention (scale bar = 20 μm); (c) [further details to be added]. Figure 5 Statistical analysis of the ratio of aggregate number to cell nucleus number in (b) of the data; express p <0.05. Detailed Implementation

[0015] This invention provides the application of chlorogenic acid in the preparation of drugs for treating neuronal intranuclear inclusion body disease.

[0016] In this invention, the concentration of chlorogenic acid is 0.4~50 μM, preferably 2~20 μM, and more preferably 10 μM; the chlorogenic acid is any one of chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid; the chlorogenic acid has a molecular glue function, which can specifically bind to and increase the affinity of polyG protein for E3 ubiquitin ligase TRIM21 protein, effectively degrading abnormally aggregated polyG protein through the ubiquitin-proteasome pathway, and reducing the load of inclusion bodies in and around the cell nucleus. The neuronal intranuclear inclusion body disease is caused by... NOTCH2NLC This is caused by abnormal amplification of the GGC gene duplication.

[0017] In this invention, the dosage form of the drug is an oral preparation.

[0018] The present invention also provides a medicament for treating neuronal intranuclear inclusion body disease, the medicament being chlorogenic acid and its pharmaceutically acceptable carrier.

[0019] In this invention, the concentration of chlorogenic acid is 0.4~50μM, preferably 2~20μM, and more preferably 10μM; the chlorogenic acid is any one of chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid.

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example

[0022] 1. Experimental Materials and Sources

[0023] Test drug: chlorogenic acid, purchased from Selleck Chemicals (S2280), purity 99.67%. Dissolved in double-distilled water under high temperature and pressure to prepare a 10 mM stock solution, stored at -20℃ for later use.

[0024] Cell line: transfected with GGC 92 HEK293 cells with -GFP repeat sequences.

[0025] Dorsal forebrain organoids: Dorsal forebrain organoids obtained by reprogramming peripheral blood mononuclear cells from healthy controls and NIID patients into human induced pluripotent stem cells (hiPSCs) and then further inducing differentiation and culture.

[0026] 2. Affinity of chlorogenic acid as a molecular glue with polyG protein and E3 ubiquitin ligase

[0027] 2.1 Calculation of binding energy of TRIM21-chlorogenic acid-polyG ternary complex

[0028] AlphaFold2 is an artificial intelligence system that can predict the three-dimensional structure of proteins with high accuracy from amino acid sequences. Inputting the amino acid sequences of polyG and TRIM21 proteins into AlphaFold2, it outputs a predicted 3D atomic model and a confidence score for each residue. The AlphaFold2 modeling output is then imported into the docking software Schrodinger Maestro. The Glide module is used to perform flexible docking of chlorogenic acid with the protein, predict the binding mode of chlorogenic acid to polyG-TRIM21, and the MM-GBSA component is used to calculate the free binding energy of Trim21-CA-polyG and the binding energy of its ternary complex. The results are shown in [Figure number missing]. Figure 1 .

[0029] Depend on Figure 1 It can be seen that the three-dimensional structure of proteins can be predicted by inputting the amino acid sequences of polyG and TRIM21 using the AlphaFold2 system, and the spatial relationship of amino acid residue pairs can be deduced using the Evoformer module. Target-based virtual screening was performed using AlphaFold2 and Schrödinger virtual screening software. The Proteinpreparation component of the molecular docking software Maestro 13.5 was used to prepare the protein files, and the LigPrep component was used to preprocess the small molecule structure files (dehydration, desalting, electron supplementation, etc., Morris, SD, et al., Identification of aStaphylococcus aureus amidase catalytic domain inhibitor to prevent biofilm formation by sequential virtual screening, molecular dynamics simulation and biological evaluation. International Journal of Biological Macromolecules, 2023. 254(Pt 2): p. 127842.). The Receptor Grid Gener component was used to define the binding site of polyG-Trim21, and the Ligand Docking component was used to perform flexible docking of chlorogenic acid with the protein. The results showed a docking score of -10.441, an XP GScore of -10.445, and a glide GScore of -10.445. Calculations using the MM-GBSA assembly indicated that chlorogenic acid binding reduced the free energy of the Trim21-polyG binary complex by 38.335 kcal / mol, confirming the stable existence of the Trim21-chlorogenic acid-polyG ternary complex.

[0030] 2.2 Cell thermal displacement experiment to verify the binding of chlorogenic acid to TRIM21

[0031] HEK293 cells were cultured in 10cm dishes. When the cell density reached 70%, they were treated with different final concentrations of chlorogenic acid (0, 10, 20, and 40 μM) for 48 hours. After trypsin collection, the cells were washed once with PBS, and then resuspended in 1mL PBS (with the corresponding concentrations of chlorogenic acid and 1× protease inhibitor added again). The cell suspension from each dish was evenly aliquoted into eight 1.5mL EP tubes (100μL of cell suspension per tube). A temperature gradient metal bath was set at 37℃, 39℃, 41℃, 43℃, 45℃, 47℃, 49℃, and 51℃. After boiling for 5 minutes, the supernatant was collected by centrifugation. Protein samples were prepared for subsequent experiments. The results are shown in [Figure number missing]. Figure 2 .

[0032] Depend on Figure 2 It can be seen that chlorogenic acid can alter the thermal stability curve of TRIM21. Figure 2 This suggests that chlorogenic acid directly binds to TRIM21 in the cellular environment.

[0033] 2.3 Surface plasmon resonance experiments to verify the interaction between chlorogenic acid and TRIM21

[0034] The Series S Sensor Chip CM5 (Cell Signaling Technology, #29104988) was assembled with the sample loading element. After pretreatment, the chip was used for sample loading. Solution preparation: Purified recombinant TRIM21 protein (Proteintech, #Ag28377) was dissolved in PBS running buffer (pH=7.4, containing 0.1% Tween 20); chlorogenic acid (Selleck, S2280) was prepared as a 20 mM solution using DMSO; 10 mM glycine-HCl buffer (pH=2.0) was used as the regeneration buffer. The specific procedure was as follows: the TRIM21 protein sample was flowed through at a flow rate of 0.5 μL / s for 600 seconds for binding; then flowed through the running buffer for 360 seconds for dissociation; finally, the sample was flowed through the regeneration buffer three times at a flow rate of 2 μL / s for 200 seconds each time. To determine binding affinity, four gradient concentrations of chlorogenic acid solution were prepared and sequentially flowed through the chip. Experimental data were collected and analyzed using the commercial SPRi analysis software (Plexera SPR Data Analysis Model, Plexera). Results are shown below. Figure 3 .

[0035] Depend on Figure 3 It is known that chlorogenic acid and TRIM21 have a dose-dependent binding (0.4 μM-50 μM), which provides biophysical evidence for their direct interaction.

[0036] 3. Therapeutic effects of chlorogenic acid in in vitro NIID cell models and organoid models

[0037] 3.1 Effect of chlorogenic acid on intracellular inclusion body load in HEK293 cells

[0038] HEK293 cells (purchased from Yuchi (Shanghai) Biotechnology Co., Ltd.) were cultured in 10cm cell culture dishes, 6-well plates, or 12-well plates with cell spreaders. When the cell density reached 60%, GGCs were transfected. 92 -GFP overexpression plasmid (from Xiangya Hospital, Central South University) was used to construct a NIID cell model. GGC was transfected. 92 After GFP treatment for 48 hours, cells were passaged at a 1:2 ratio and allowed to adhere overnight. Then, cells were treated with chlorogenic acid at a final concentration of 10 μM for 48 hours. Cells in 10 cm cell culture dishes were used for protein extraction, and protein immunoprecipitation was performed using GFP antibody. Cells in 6-well plates were treated with 10 μM MG132 8 hours before sample collection to inhibit proteasome function. Cells on slides in 12-well plates were used for immunofluorescence detection. Results are shown below. Figure 4 .

[0039] Depend on Figure 4 It can be seen that HEK293 cells transfected with polyG 92 -GFP followed by perinuclear or intranuclear inclusions suggests that the model can effectively simulate NIID-related pathological features. Figure 4 (ab in the expression GGC). 92 In the HEK293 cell NIID model of -GFP, the protein immunoprecipitation results showed that after 48 hours of treatment with 10 μM chlorogenic acid, the levels of polyG protein monomers and the polyG inclusion body load in the input were significantly reduced. Figure 4 In the ce), and the GFP ubiquitination level was significantly increased ( Figure 4 (c) Furthermore, after MG132 intervention, both polyG protein monomers and polyG inclusion bodies increased, and the degradation effect of chlorogenic acid on polyG disappeared. Figure 4 (f in the text). Immunofluorescence results further confirmed that chlorogenic acid treatment can significantly reduce the polyG inclusion body load (f in the text). Figure 4 (gh in the text). The above results collectively indicate that chlorogenic acid can degrade the polyG abnormal protein and its inclusion bodies through molecular glue interactions.

[0040] 3.2 Effect of Chlorogenic Acid Addition on Inclusion Body Load in HiPSC-Derived Brain Organoids from NIID Patients

[0041] Following the instructions for the dorsal forebrain differentiation induction kit (STEMCELL, #08620), dorsal forebrain organoids derived from hiPSCs of NIID patients were induced and differentiated. The specific steps were as follows: Single-cell hiPSC suspensions were seeded into AggreWell™ 800-well plates, 10,000 cells per well, followed by centrifugation. After culturing in Formation Medium with half-daily medium changes for 5 days, embryoid bodies (EBs) were collected using a 37μm cell sieve and transferred to ultra-low adsorption 6-well plates, seeding 25-40 EBs per well. Cultured in Expansion Medium, with medium changes every 48 hours, until day 25, when the medium was replaced with Differentiation Medium. From day 43, organoids were transferred to Maintenance Medium, with medium changes every 2-3 days; after day 50, the culture density was adjusted to less than 10 organoids per well, and the culture volume was increased to 3 mL per well. Chlorogenic acid was added to the culture medium at 70 days to bring the final concentration to 10 μM. Seven days later, samples of the dorsal forebrain organoids were collected for frozen section immunofluorescence analysis. Results are shown below. Figure 5 .

[0042] Depend on Figure 5 It is known that hiPSCs derived from NIID patients can be successfully induced to differentiate into dorsal forebrain organoids. Figure 5 (a) After culturing for 70 days, the addition of 10 μM chlorogenic acid to the culture medium for 7 days resulted in a significant reduction in inclusion bodies in the dorsal forebrain organoids observed by immunofluorescence. Figure 5 (bc in the middle).

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of chlorogenic acid in the preparation of drugs for treating neuronal intranuclear inclusion body disease.

2. The application according to claim 1, characterized in that, The concentration of chlorogenic acid is 0.4~50 μM.

3. The application according to claim 1, characterized in that, The neuronal intranuclear inclusion body disease is caused by NOTCH2NLC This is caused by abnormal amplification of the GGC gene duplication.

4. The application according to claim 1, characterized in that, The drug is an oral formulation.

5. The application according to claim 1, characterized in that, The chlorogenic acid is any one of the following: chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid.

6. A pharmaceutical agent for treating neuronal intranuclear inclusion body disease, characterized in that, The agent is chlorogenic acid and its pharmaceutically acceptable carrier.

7. The pharmaceutical preparation according to claim 6, characterized in that, The chlorogenic acid is any one of the following: chlorogenic acid monomer, pharmaceutically acceptable salt of chlorogenic acid, or plant extract containing chlorogenic acid.

8. The pharmaceutical preparation according to claim 6, characterized in that, The concentration of chlorogenic acid is 0.4~50 μM.

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