Application of parecoxib as MEF2A transcription inhibitor in preparation of antiepileptic drugs

Abstract

The invention relates to the technical field of neuroscience and drugs, and discloses application of parecoxib as an MEF2A transcription inhibitor in preparation of antiepileptic drugs, which is characterized in that the parecoxib is in targeted combination with N-terminal alpha1 helix and MADS box structural domain of MEF2A protein to inhibit DNA binding capacity of MEF2A, so that downstream NEK7 / CD74 gene transcription is blocked, and microglial cell mediated neuroinflammation is reduced; according to the present invention, the transcriptional activity action mechanism of parecoxib is inhibited through the MEF2A targeting DNA binding interface, specifically, the parecoxib is specifically bound with the N-terminal alpha 1 helix (residues ARG17, THR20, PHE21, LYS23 and ARG24) and MADS box structure domain of MEF2A so as to inhibit the conformational change and the DNA binding ability (the molecular docking simulation shows the high affinity binding), and the transcription of the downstream target gene (such as NEK7 and CD74) is inhibited by blocking the binding of the MEF2A and the DNA so as to inhibit the transcriptional activity of the MEF2A; therefore, the activation of NLRP3 inflammasomes is reduced; excessive activation of microglial cells is inhibited, and the steady state of the neural network is maintained.

Description

Technical Field

[0001] This invention relates to the fields of neuroscience and drug development technology, specifically the application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs. Background Technology

[0002] Currently, epilepsy treatment mainly relies on antiepileptic drugs (such as benzodiazepines and sodium valproate) to inhibit excessive neuronal discharge, but there is a lack of targeted intervention for epilepsy-related neuroinflammation and microglia-mediated pathological synaptic remodeling. Studies have shown that ATP release triggered by epileptic seizures activates microglia's P2X7 receptor, triggering MEF2A-dependent inflammatory pathways (such as NLRP3 inflammasome activation), exacerbating neuronal hyperexcitability and loss of synaptic inhibitory function. However, there are currently no corresponding MEF2A inhibitors, making it impossible to effectively intervene against this target. Parecoxib is a known cyclooxygenase (COX) inhibitor with well-established human safety data, but its transcriptional regulatory function targeting MEF2A has not yet been discovered. Therefore, this application proposes the application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs. Summary of the Invention

[0003] The purpose of this invention is to provide the application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs, so as to solve the problems mentioned in the background art.

[0004] Parecoxib, originally a cyclooxygenase (COX) inhibitor commonly used in anti-inflammatory therapy, is the first drug to specifically target the MEF2A protein and inhibit its transcriptional activity. MEF2A (myotrophic factor 2A) is a transcription factor that plays a crucial role in epilepsy-related neuroinflammation: during a seizure, ATP release activates the P2X7 receptor on microglia, triggering MEF2A-dependent inflammatory pathways (such as NLRP3 inflammasome activation), leading to neuronal hyperexcitability and synaptic dysfunction. Existing antiepileptic drugs (such as benzodiazepines) only inhibit neuronal firing but cannot specifically address MEF2A-mediated inflammation. This application utilizes parecoxib to block the DNA binding ability of MEF2A, inhibiting the transcription of downstream genes (NEK7 and CD74), thereby reducing microglia-mediated neuroinflammation and maintaining neural network homeostasis. The ultimate goal is to develop novel antiepileptic drugs applicable to neurological diseases such as epilepsy, Alzheimer's disease, and Parkinson's disease.

[0005] In a first aspect, to achieve the above objectives, the present invention provides the following technical solution: the application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs, comprising: Parecoxib inhibits the DNA binding ability of MEF2A by targeting and binding to the N-terminal α1 helix and MADS box domain of the MEF2A protein, thereby blocking the transcription of downstream NEK7 / CD74 genes and reducing microglia-mediated neuroinflammation.

[0006] The N-terminal α1 helix is ​​one of residues ARG17, THR20, PHE21, LYS23, and ARG24.

[0007] The effective in vitro concentration of parecoxib is 80 μM.

[0008] The in vivo dosage of parecoxib is 100 μM / 2 μL per side via intraventricular injection.

[0009] The binding affinity of parecoxib to MEF2A is ≤-8.5 kcal / mol.

[0010] Parecoxib reduced the binding affinity of MEF2A to the NEK7 promoter from 3.9 times to ≤1.5 times, and to the CD74 promoter from 2.2 times to ≤1.3 times.

[0011] Secondly, to achieve the above objectives, the present invention provides the following technical solution: an antiepileptic drug composition comprising a therapeutically effective amount of parecoxib and a pharmaceutically acceptable carrier.

[0012] The antiepileptic drug composition inhibits the MEF2A-dependent inflammatory pathway, thereby reducing NLRP3 inflammasome activation and IL-1β mRNA expression by ≥40%.

[0013] Parecoxib is administered to brain tissue via local drug delivery. Injection is preferred in the ventricles or hippocampus.

[0014] The carrier is the AAV-shMef2a carrier.

[0015] Compared with the prior art, the beneficial effects of the present invention are: In this invention, parecoxib inhibits the transcriptional activity of MEF2A by targeting the DNA binding interface of MEF2A. Specifically, parecoxib specifically binds to the N-terminal α1 helix (residues ARG17, THR20, PHE21, LYS23, ARG24) and MADS box domain of MEF2A, inhibiting its conformational changes and DNA binding ability (molecular docking simulation shows high affinity binding). By blocking the binding of MEF2A to DNA, it inhibits the transcription of its downstream target genes (such as NEK7 and CD74), thereby reducing NLRP3 inflammasome activation; inhibiting excessive activation of microglia, and maintaining neural network homeostasis. Attached Figure Description

[0016] Figure 1 This is a simulation diagram of molecular docking of the present invention (MEF2A binding with parecoxib); Figure 2 This is a schematic diagram illustrating the effect of parecoxib on BV2 cell viability during the MTT assay of this invention. Figure 3 This is a schematic diagram illustrating the effect of parecoxib on the viability of HEK293 cells during the MTT assay of this invention. Figure 4 This is an immunostatistical graph illustrating the inhibition of MEF2A-mediated inflammatory response in this invention. Figure 5 This is an immunofluorescence image of the inhibition of MEF2A-mediated inflammatory response in this invention; Figure 6 This is a schematic diagram of the Western blot analysis of MEF2A protein expression according to the present invention; Figure 7 This is a bar chart showing the expression of MEF2A, CD74, NEK7, and GAPDH proteins under Western blot analysis according to the present invention. Figure 8 This is a schematic diagram showing the occupancy of MEF2A in the NEK7 promoter region of BV2 cells in the ChIP-qPCR results of this invention; Figure 9 This is a schematic diagram showing the occupancy of MEF2A in the CD74 promoter region of BV2 cells in the ChIP-qPCR results of this invention; Figure 10 This is a schematic diagram showing the binding of MEF2A to the NEK7 promoter in the ChIP-qPCR results of this invention; Figure 11 This is a schematic diagram showing the binding of MEF2A to the CD74 promoter in the ChIP-qPCR results of this invention; Figure 12 This is a line graph showing the frequency of epileptic seizures in the mouse model of epilepsy of this invention; Figure 13 The electroencephalogram waveforms and spectrograms for EEG discharge analysis in the epileptic mouse model of this invention; Figure 14 This is an image of IBA1 staining in the mouse model of epilepsy of this invention; Figure 15 This is a line graph showing the IBA1 staining analysis in the epilepsy mouse model of this invention. Figure 16 This is a Nissl staining image of the CA3 region in a hippocampal slice from a mouse model of epilepsy according to the present invention. Figure 17 This is a stained bar chart of the epilepsy mouse model of the present invention; Figure 18 This is a swimming trajectory diagram of the Morris water maze test in the epilepsy mouse model of the present invention; Figure 19 This is a line graph of the escape latency in the epilepsy mouse model of the present invention; Figure 20 This is a bar chart showing the number of platform crossings in the epilepsy mouse model of the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In a first aspect, to achieve the above objectives, the present invention provides the following technical solution: the application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs, comprising: Parecoxib targets and binds to the N-terminal α1 helix of the MEF2A protein (the N-terminal α1 helix is ​​one of residues ARG17, THR20, PHE21, LYS23, and ARG24) and the MADS box domain, inhibiting its conformational changes and DNA binding ability (molecular docking simulations show high affinity binding). By blocking the binding of MEF2A to DNA, it inhibits the transcription of its downstream target genes (such as NEK7 and CD74), thereby: reducing NLRP3 inflammasome activation; inhibiting microglial overactivation; and maintaining neural network homeostasis.

[0019] The N-terminal α1 helix of the MEF2A protein (the N-terminal α1 helix can be one of residues ARG17, THR20, PHE21, LYS23, or ARG24) and the MADS box domain are responsible for the conformational changes and DNA binding of MEF2A. Parecoxib locks key residues of the α1 helix of MEF2A (such as LYS23, ARG24, and THR20) through hydrogen bonding and hydrophobic interactions, inhibiting its conformational transition from "closed" to "open". Molecular docking simulations show a binding affinity ≤ -8.5 kcal / mol (high affinity), indicating strong binding. After binding, MEF2A cannot bind to the DNA promoter region, thus losing transcriptional activity.

[0020] The effective in vitro concentration of parecoxib is 80 μM; the in vivo dosage of parecoxib is 100 μM / 2 μL per side via intracerebrospinal injection; the binding affinity of parecoxib to MEF2A is ≤-8.5 kcal / mol; parecoxib reduces the binding affinity of MEF2A to the NEK7 promoter from 3.9 times to ≤1.5 times, and to the CD74 promoter from 2.2 times to ≤1.3 times.

[0021] Secondly, to achieve the above objectives, the present invention provides the following technical solution: an antiepileptic drug composition comprising a therapeutically effective amount of parecoxib and a pharmaceutically acceptable carrier, wherein the carrier is an AAV-shMef2a carrier; the antiepileptic drug composition inhibits the MEF2A-dependent inflammatory pathway, thereby reducing NLRP3 inflammasome activation and IL-1β mRNA expression by ≥40%.

[0022] Parecoxib is administered to brain tissue via local administration; preferably by injection into the ventricle or hippocampus.

[0023] Parecoxib, as a MEF2A transcriptional inhibitor, is used in the preparation of antiepileptic drugs. The inhibitor is used to treat neurological diseases caused by abnormalities in the P2X7 receptor-ATP-MEF2A signaling axis, including epilepsy, Alzheimer's disease, and Parkinson's disease.

[0024] In vitro and in vivo efficacy verification of parecoxib; In vitro experiments: In BV2 microglia, 80 μM parecoxib significantly inhibited ATP-induced MEF2A protein expression (p<0.01) and downstream NEK7 / CD74 (p<0.05). ChIP-qPCR confirmed that parecoxib reduced the binding of MEF2A to the promoter regions of NEK7 (from 3.9-fold to 1.5-fold, p<0.01) and CD74 (from 2.2-fold to 1.3-fold, p<0.001).

[0025] In vivo experiments: In a KA-induced epilepsy mouse model, intraventricular injection of parecoxib or hippocampal AAV-shMef2a-mediated MEF2A knockdown significantly reduced MEF2A levels, suggesting that parecoxib's inhibitory effect on MEF2A can reach the level of adenovirus. Intraventricular injection of parecoxib or hippocampal AAV-shMef2a-mediated MEF2A knockdown reduced the frequency of chronic phase epileptic seizures (p<0.01), alleviated the severity of abnormal EEG discharges (p<0.05), and decreased the mRNA level of the microglia-mediated neuroinflammatory factor IL1β.

[0026] The synergistic therapeutic advantages of parecoxib: In addition to its MEF2A inhibitory effect, its COX-2 inhibitory properties can further reduce prostaglandin-mediated neuroinflammation and enhance the anti-epileptic efficacy.

[0027] Experimental Example 1: Verification of the MEF2A inhibitory activity of parecoxib: Step 1: Molecular docking and binding energy analysis: The experimental method was as follows: AutoDock Vina software was used to simulate the binding site of parecoxib and MEF2A, confirming that residues LYS23, ARG24, and THR20 were key binding residues, with a binding energy of -8.5 kcal / mol (high affinity). Figure 1 As shown in the figure, the molecular docking simulation (MEF2A binding with parecoxib) shows that parecoxib (molecular structure) binds to the N-terminal α1 helix of the MEF2A protein. Key residues include LYS23, ARG24 and THR20. This confirms that parecoxib specifically binds to MEF2A and inhibits its conformational changes and DNA binding ability.

[0028] Step 2: Cytotoxicity test: The experimental method was as follows: cell viability was detected by MTT assay, concentration range 5-100 μM (dissolved in PBS solution); the MTT assay showed that parecoxib had no significant toxicity to BV2 and HEK293 cells at ≤100 μM (survival rate >95%), and was still safe at a concentration of 80 μM (p>0.05). Figure 2 and 3 (as shown) Figure 2 This is a schematic diagram illustrating the effect of parecoxib on BV2 cell viability during the MTT assay. Figure 3 The diagram shows the cell viability of parecoxib in HEK293 cells during the MTT assay. As can be seen from the figure, the cell viability was >95% when parecoxib was ≤10μM and it was still safe at 80μM (p>0.05), which proves its low toxicity.

[0029] Step 3: Inhibition of MEF2A-mediated inflammatory response: The experimental method was as follows: BV2 cells were stimulated with ATP (800 μM), treated with parecoxib for 24 h, and iNOS was detected by immunofluorescence staining; after ATP (800 μM) stimulation of BV2 cells, 80 μM parecoxib significantly reduced iNOS expression (p<0.05). Figure 4 and 5 As shown in the figure, this confirms the inhibition of the inflammatory response; Figure 4 To suppress the immunostatistical map in the MEF2A-mediated inflammatory response, Figure 5 Immunofluorescence images were used to suppress MEF2A-mediated inflammatory responses, demonstrating iNOS expression levels after ATP stimulation. Figure 4 statistics Figure 5The mean fluorescence intensity of INOS in each group of cells was measured. The t-test was used to compare two groups, and one-way ANOVA was used to compare multiple groups. ns p > 0.05, *p<0.05, **p<0.01, ***p<0.001 vs comparison group; Specifically, while stimulating the Bv2 cell line with ATP (simulating epilepsy-like activity concentration), parecoxib was added to the culture medium, resulting in final concentrations of parecoxib in the culture medium of 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, and 100 μM. After treating Bv2 cells with PBS containing parecoxib and ATP for 24 h, the expression level of iNOS was detected by immunofluorescence staining. Images were acquired using a confocal microscope (scale bar = 20 μm). The average fluorescence intensity statistics of iNOS are shown in […]. Figure 4 .

[0030] Step 4: Western blot analysis: The experimental method was as follows: Western blots were performed in the control group, ATP treatment group, and ATP + parecoxib group. In BV2 microglia, 80 μM parecoxib significantly inhibited ATP-induced MEF2A protein expression (p<0.01) and downstream NEK7 / CD74 expression (p<0.05). Figure 6 and Figure 7 (as shown) Figure 6 This is a schematic diagram of Western blot analysis of MEF2A protein expression. The image shows a white blot image, displaying the expression of MEF2A, NEK7, CD74, and GAPDH. Figure 7 The bar chart shows the expression of MEF2A, CD74, NEK7, and GAPDH proteins under Western blot analysis. Western blot analysis was performed on the expression of MEF2A, CD74, NEK7, and GAPDH proteins in BV2 cells from the control group, ATP(e) treatment group, and ATP(e) + parecoxib treatment group. The figure shows that parecoxib inhibited ATP-induced MEF2A protein expression (p<0.01) and downstream NEK7 / CD74 expression (p<0.05).

[0031] Step 5: ChIP-qPCR Results: Experimental Method: Chromatin immunoprecipitation (ChIP) followed by qPCR quantification; ChIP-qPCR confirmed that parecoxib reduced the binding of MEF2A to the promoter regions of NEK7 (from 3.9-fold to 1.5-fold, p<0.01) and CD74 (from 2.2-fold to 1.3-fold, p<0.001) (e.g., ...). Figures 8-11 (as shown) Figure 8This is a schematic diagram showing the occupancy of MEF2A in the NEK7 promoter region of BV2 cells in ChIP-qPCR results. Chromatin immunoprecipitation (ChIP) analysis confirmed that MEF2A occupies the NEK7 promoter region of BV2 cells. Figure 9 This is a schematic diagram showing the occupancy of MEF2A in the CD74 promoter region of BV2 cells in the ChIP-qPCR results. Chromatin immunoprecipitation (ChIP) analysis confirmed that MEF2A occupies the CD74 promoter region of BV2 cells. Figure 10 This is a schematic diagram of the binding of MEF2A to the NEK7 promoter in ChIP-qPCR results. The binding of MEF2A to the NEK7 promoter was detected by chromatin immunoprecipitation (ChIP) in BV2 cells treated with parecoxib. Figure 11 This is a schematic diagram of the binding of MEF2A to the CD74 promoter in ChIP-qPCR results. The binding of MEF2A to the CD74 promoter was detected by chromatin immunoprecipitation (ChIP) in BV2 cells treated with parecoxib.

[0032] Experimental Example 2: In vivo efficacy evaluation: Step 1: Establishment of the epilepsy model: The epilepsy mouse model (KA-3d model) was induced by KA (kamarinic acid) injection.

[0033] Step 2: Parecoxib intervention program, group setup: Control group: sham surgery + saline; KA group: KA injection + saline; Experimental group 1: KA injection + intraventricular parecoxib (100μM, 2 μL / side, icv); Experimental group 2: KA injection + AAV-shMef2a (microglia-specific knockdown of MEF2A in the CA3 region of the hippocampus).

[0034] Step 3: Efficacy evaluation indicators: Behavioral studies: Video monitoring of seizure frequency and duration (e.g.) Figure 12 (as shown) Figure 12 Line graphs showing the seizure frequency in a mouse model of epilepsy, displaying the daily seizure frequency in the KA group, AAV-shMEF2A+KA group, and parecoxib+KA group; as can be seen from the graph, parecoxib significantly reduced the seizure frequency (p<0.01).

[0035] Electrophysiology: Electroencephalography (EEG) analysis of epileptiform discharge waveforms (e.g.) Figure 13 (as shown) Figure 13 EEG waveforms and spectrograms were analyzed in a mouse model of epilepsy. The figures show that parecoxib reduced the severity of abnormal discharges (p<0.05).

[0036] Pathology: IBA1 staining confirmed the degree of microglial activation after parecoxib treatment (e.g. Figure 14 and Figure 15 As shown), Nissl staining was used to detect the degree of neuronal damage in the mouse hippocampus (e.g. Figure 16 and Figure 17 (as shown) Figure 14 The image shows IBA1 staining in a mouse model of epilepsy. IBA1 immunostaining was performed on the morphology of microglia in the hippocampus of the KA group, AAV-shMEF2A+KA group, and parecoxib+KA group. Scale bar: 20 micrometers. Sholl analysis was then performed to assess the complexity of microglia branching. Figure 15 Line graph showing IBA1 staining analysis in a mouse model of epilepsy; Figure 16 Nissl staining images of the CA3 region in hippocampal sections from a mouse model of epilepsy. The images were taken from the sham-operated group, KA-3d group, AAV-shMEF2A KA-3d group, and par+KA-3d group. Quantitative analysis was performed on the Nissl staining positive cells in the CA3 region of the hippocampal sections. Figure 17 The image shows a stained bar graph in a mouse model of epilepsy. As can be seen from the graph, parecoxib reduces microglia activation, decreases IL-1β mRNA, and alleviates neuronal damage.

[0037] Cognitive function: Morris water maze assesses spatial memory (e.g.) Figure 18 , Figure 19 and Figure 20 (as shown) Figure 18 The swimming trajectory diagrams for the Morris water maze test in a mouse model of epilepsy are obtained from the training experiments in the KA group, AAV-shMEF2A+KA group, and parecoxib+KA group. Figure 19 A line graph of escape latency in a mouse model of epilepsy; Figure 20 The bar chart shows the number of platform crossings in a mouse model of epilepsy; the Morris water maze performance (escape latency and number of platform crossings) in the KA group, AAV-shMEF2A+KA group, and parecoxib+KA group; the figure shows that parecoxib improves spatial memory, demonstrating the recovery of cognitive function.

[0038] In summary, parecoxib in this application inhibits the transcriptional activity of MEF2A by targeting the DNA binding interface of MEF2A. Specifically, parecoxib specifically binds to the N-terminal α1 helix (residues ARG17, THR20, PHE21, LYS23, ARG24) and MADS box domain of MEF2A, inhibiting its conformational changes and DNA binding ability (molecular docking simulation shows high affinity binding). By blocking the binding of MEF2A to DNA, it inhibits the transcription of its downstream target genes (such as NEK7 and CD74), thereby reducing NLRP3 inflammasome activation; inhibiting excessive microglial activation, and maintaining neural network homeostasis.

[0039] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs, characterized in that, include: Parecoxib inhibits the DNA binding ability of MEF2A by targeting and binding to the N-terminal α1 helix and MADS box domain of the MEF2A protein, thereby blocking the transcription of downstream NEK7 / CD74 genes and reducing microglia-mediated neuroinflammation.

2. The use of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs according to claim 1, characterized in that: The N-terminal α1 helix is ​​one of residues ARG17, THR20, PHE21, LYS23, and ARG24.

3. The use of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs according to claim 1, characterized in that: The effective in vitro concentration of parecoxib is 80 μM.

4. The use of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs according to claim 1, characterized in that: The in vivo dosage of parecoxib is 100 μM / 2 μL per side via intraventricular injection.

5. The use of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs according to claim 1, characterized in that: The binding affinity of parecoxib to MEF2A is ≤-8.5 kcal / mol.

6. The use of parecoxib as a MEF2A transcriptional inhibitor in the preparation of antiepileptic drugs according to claim 1, characterized in that: Parecoxib reduced the binding affinity of MEF2A to the NEK7 promoter from 3.9 times to ≤1.5 times and to the CD74 promoter from 2.2 times to ≤1.3 times.

7. An antiepileptic drug composition, characterized in that: It contains a therapeutically effective amount of parecoxib and a pharmaceutically acceptable carrier.

8. The antiepileptic drug composition according to claim 7, characterized in that: The antiepileptic drug composition reduces NLRP3 inflammasome activation and IL-1β mRNA expression by ≥40% by inhibiting the MEF2A-dependent inflammatory pathway.

9. The antiepileptic drug composition according to claim 7, characterized in that: Parecoxib is administered to brain tissue via local drug delivery. Injection is preferred in the ventricles or hippocampus.

10. The antiepileptic drug composition according to claim 7, characterized in that: The carrier is the AAV-shMef2a carrier.

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