KR102240928B1 - Pharmaceutical composition for prevention or treatment of central nervous system diseases by TDP-43 mediated stress granule aggregation in cells via inhibiting ATXN2 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating central nervous system diseases caused by the generation of stress granules in cells, and more particularly, to a pharmaceutical composition for preventing and treating central nervous system diseases, which comprises any one selected from the group consisting of Epiandrosterone, Topiramate and pharmaceutically acceptable salts thereof as an active ingredient to inhibit ATXN2 and thus suppress abnormal aggregation of TDP-43 protein, thereby achieving the effect of remarkably lowering the production of stress granules in amyotrophic lateral sclerosis and frontal dementia cells which are the central nervous system diseases, and thus can be advantageously used for developing the preventive and therapeutic agents for amyotrophic lateral sclerosis or frontal dementia.

Description

Pharmaceutical composition for prevention or treatment of central nervous system diseases by TDP-43 mediated stress granule aggregation in cells via inhibiting ATXN2}

The present invention relates to a composition for preventing and treating central nervous system diseases, and more particularly, to a composition for preventing and treating central nervous system diseases that inhibits overproduction of TDP-43 protein by controlling ATXN2, a modulator of stress granules.

The accumulation of abnormal proteins such as RNA/DNA binding protein TDP-43 and stress granules and their mutations is a type of early dementia, with pretemporal dementia or frontotemporaldementia (FTD) and motility associated with speech and behavioral disorders. It has been found in amyotrophic lateral sclerosis (ALS), which causes disorders, and interest in the association between these proteins and brain diseases is increasing.

TAR DNA-binding protein 43 (TDP-43) is involved in the exon splicing of various genes, including the survival motor neuron (SMN) gene, which is related to motor neuron disease. It is a key protein that contributes to the generation of aggregated masses of proteins, RNA, DNA, etc., collectively referred to as stress granules when cells are placed in a stressful situation due to factors. In addition, as a protein encoded by the TAR DNA-binding protein gene, it regulates mRNA safety, transport, and local translation in neurons in the central nervous system. As a cause of overproduction of TDP-43, dysfunction-related diseases are caused by several gene mutations. In particular, a change in the DNA sequence of the TDP-43 gene causes the malfunction of gene self-regulation, resulting in overexpression of the protein. And other genes related to frontal dementia or amyotrophic lateral sclerosis, and it was found to be essential for the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it has been reported that abnormal aggregates such as stress granules are found in about 90-95% of ALS patients.

Ataxin 2 is an RNA-binding protein that acts as a regulator of stress granules and has multiple roles in RNA metabolism. In general, it has been known that aggregated proteins such as TDP-43 in stress granules induce the formation of pathological protein aggregates in amyotrophic lateral sclerosis (ALS), and aggregation of granules improves the level of insoluble TDP-43 and dissolves stress granules. It was later reported that the TDP-43 inclusion could persist.

In addition, Korean Patent Registration No. 10-1924269 discloses that ALS is related to ubiquitin-proteosome system (UPS) damage, and that UPS damage is closely related to TDP-43/ATXN2. It has been shown that inclusion formation can be directly involved as a biomarker for Alzheimer's and neurodegenerative disorders.

However, since the original role of TDP-43 is involved in the growth and maintenance of cells, direct inhibition of TDP-43 acts lethal to the cells, so that ataxin-2 (ATXN2) is involved in the regulatory signaling pathway of TDP-43. Although research is being conducted in related academic circles, ATXN2 is only presented as a pathological mechanism for treating ALS and FTD, and despite various studies, effective treatment of ALS and FTD by effective inhibition of ATXN2 is difficult. Therefore, in the present invention, epiandrosterone and topiramate, which are binding substances that can effectively inhibit ATXN2, are selected using an artificial intelligence-based binding degree prediction technique, and the effect of remarkably reducing the formation of stress granules by them is confirmed to prevent and treat ALS and FTD. It is expected to be able to discover new drug candidates for

Accordingly, the present inventors selected epiandrosterone and topiramate, which are binding substances capable of effectively inhibiting ATXN2, through a drug-protein interaction prediction algorithm, and inhibited the formation of stress granules due to overexpression of TDP-43 for these two substances, resulting in amyotrophic properties. As a candidate material for effective treatment for lateral sclerosis and frontal dementia disease, the effect was confirmed as an experiment, and the present invention was completed.

1. Korean Patent Registration No. 10-1924269, Method for the prevention, alleviation or treatment of amyotrophic lateral sclerosis, registration date November 26, 2018 2. US Patent No. US09958460, Biomarkers and diagnostic methods for Alzheimer's disease and other neurodegenerative disorders, registration date May 1, 2018

An object of the present invention is to inhibit the abnormal aggregation of TDP-43 by inhibiting ATXN2 containing any one selected from the group consisting of Epiandrosterone, Topiramate, and pharmaceutically acceptable salts thereof as an active ingredient, thereby inhibiting the abnormal aggregation of TDP-43, thereby inhibiting the stress granules of cells. granule) to provide a pharmaceutical composition for the prevention and treatment of central nervous system diseases caused by the generation.

In order to achieve the above object, the present invention inhibits ATXN2 containing any one selected from the group consisting of Epiandrosterone, Topiramate and their pharmaceutically acceptable salts as an active ingredient, thereby inhibiting abnormal aggregation of TDP-43 It provides a pharmaceutical composition for the prevention and treatment of central nervous system diseases caused by the generation of stress granules.

In the present invention, the central nervous system disease may be amyotrophic lateral sclerosis or frontal lobe dementia.

The pharmaceutical composition for the prevention and treatment of central nervous system diseases caused by the generation of stress granules in cells according to the present invention significantly reduces the generation of stress granules by inhibiting ATXN2 and inhibiting abnormal aggregation of TDP-43. Through the effect, it can be usefully used in the development of a treatment and prevention of amyotrophic lateral sclerosis or frontal dementia.

1 is a graph showing the results of a stress granule generation experiment when oxidative stress is applied to a TDP43-tGFP U2O2 stable cell line and treated with 1 μM of epiandrosterone or 1 μM of topiramate.
2 is a graph showing the results of an experiment in the ratio of cells without aggregated granules to the total number of cells when stress granules due to TDP-43 protein aggregation are generated.

Hereinafter, the specific content of the invention will be described in detail.

The present invention inhibits ATXN2 containing any one selected from the group consisting of Epiandrosterone, Topiramate, and their pharmaceutically acceptable salts as an active ingredient, thereby inhibiting abnormal aggregation of TDP-43, thereby inhibiting the stress granule of cells. It provides a pharmaceutical composition for the prevention and treatment of central nervous system diseases caused by the production.

The central nervous system disease of the present invention is not particularly limited as long as it is a central nervous system disease caused by the generation of stress granules of cells, but may preferably be amyotrophic lateral sclerosis or frontal lobe dementia.

The central nervous system disease, amyotrophic lateral sclerosis, is caused by mutation of a gene called superoxide dismutase-1 (SOD1) or nerve damage of immune cells called microglial cells and astrocytes. Microglial cells are caused by cell death caused by genetic programs, and motor neurons are destroyed due to blockage or deficiency of the supply of neurotrophic factors.

In addition, frontal dementia, which is a central nervous system disease, is caused by a hereditary or spontaneous disorder (a disorder that occurs for unknown reasons) due to degeneration of the frontal and temporal lobes of the brain, and the size of the frontal and temporal lobes is reduced and nerve cells are reduced. It is a cranial nerve disease in which mental functions such as memory, thinking, judgment, and learning ability gradually decline due to loss of energy.

The present invention was completed by identifying new uses of Epiandrosterone, Topiramate and their pharmaceutically acceptable salts for central nervous system diseases, wherein Epiandrosterone is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain and gastrointestinal tract, and It functions to enhance or regulate brain function, and when deficient, it is an organic brain syndrome, showing symptoms of memory impairment, confusion, and delirium, and is associated with diseases related to the brain such as depression, irritability and judgment disorder. The Topiramate increases the GABAa-mediated inhibitory impulse (GABAa-mediated inhibitory impulse) through GABA (γ-aminobutyric acid) and inhibits α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and the kinate glutamate receptor. It is an anticonvulsant drug that has a variety of mechanisms, such as a function of blocking and regulating various ion channels, and is used as an antiepileptic drug in treatment related to brain diseases. Thus, the known pharmaceutical uses of Epiandrosterone and Topiramate are irrelevant to the present invention.

The present invention uses a Molecule transformer Drug Target Interaction (MT-DTI) deep learning artificial intelligence model (Shin et al., 2019) that predicts drug-protein binding capacity to regulate TDP-43 protein and bind to ATXN2. Epiandrosterone and Topiramate were selected as substances, and the drug-protein interaction prediction algorithm (MT-DTI, Molecule Transformer Drug Target Interaction) in the present invention is expensive and time consuming in the existing DTI (Drug Target Interaction) prediction model. As a new drug-protein interaction prediction algorithm model, MT-DTI effectively models complex chemical structures by reflecting the self-attention mechanism in the model to overcome the insignificant limit in the case that candidate substances derived from the program and lead to actual new drugs are insignificant. And by using PubChem's 97 million compound database for preliminary learning, the precision within the program for predicting drug-protein interactions was improved.

Specifically, Molecule transformer Drug Target Interaction (MT-DTI) deep learning artificial intelligence model (Shin et al. , 2019), the substances identified in the Molecule transformer Drug Target Interaction (MT-DTI) deep learning artificial intelligence model were selected for small molecule compounds published in PubChem, ChEMBL, DrugBank, etc.

After that, based on the derived substances, the transcriptome data set for each substance treatment of the connectivity map (CMAP) was analyzed and compared (Subramanian et al., 2017), and the result was confirmed on the binding score binding to ATXN2 and the RNA expression profile. Epiandrosteron and topiramate were selected.

In addition, the selected epiandrosteron and topiramate inhibited ATXN2 and inhibited abnormal aggregation of TDP-43, thereby confirming the preventive and therapeutic effects of central nervous system diseases caused by the generation of stress granules in cells. TDP43-tGFP U2O2 stable cell line (TDP43-tGFP U2O2 stable cell line), a cell line that can overexpress TDP43 in the receiving situation to form stress granules, and characteristically observe the stress granules through a fluorescence microscope due to the fluorescent protein (GFP) fused to the TDP-43 protein. Innoprot, Spain), when stress granules due to TDP-43 protein aggregation induced by oxidative stress were generated and produced, it can be confirmed through the ratio of cells without aggregated granules to the total number of cells. It is apparent that the effect of the pharmaceutical composition according to the present invention is described, and the present invention is not limited by the examples described below.

<Example: Confirmation of the effect of epiandrosterone and topiramate>

TDP43-tGFP U2O2 stable cell line (Innoprot, Spain) was used, and 10% fetal bovine serum and 10 mg/mL puromycin, 50 mg/in DMEM/Nutrient mixture F-12 Ham (Sigma-Aldrich) for initial cell proliferation. Using a nutrient medium (complete growth medium) containing mL hygromycin was incubated for 24 hours at a temperature of 37°C and in an environment of 5% carbon dioxide.

At about 90% confluency density confirmed by a microscope, 10,000 cells were dispensed into each well of a 96-well plate. At this time, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was treated at a concentration of 5 mM and cultured for 16-24 hours to induce the expression of TDP-43 protein. While exchanging the culture medium in which the cells were cultured, 10 μM arimoclomol as a positive control, 1 μM epiandrosterone, and 1 μM topiramate were treated as a positive control, and a well containing only cells without any treatment was used as a negative control (positive control: 10 μM). arimoclomol, negative control: untreated TDP43-tGFP U2O2 stable cell line, experimental group: 1 μM epiandrosterone, 1 μM topiramate).

Each was incubated for 30 minutes, and after 30 minutes, the existing culture medium was removed and the culture medium containing 250 μM sodium arsenite was treated to create an environment in which oxidative stress was formed in the cells.

After completing the treatment of the test substance on the cells, aggregation of the fluorescent protein (GFP) and stress granules was confirmed through a fluorescence microscope, and the results are shown in Table 1 and FIG. 1.

In addition, the number of individuals not formed by the stress granules was checked to calculate the ratio of stress granule free cells/total cells, and the results are shown in Table 2 and FIG. 2.

All experiments were conducted by preparing 3 wells for each experimental group, and accordingly, through 3 repetitions, a total of 9 measurement values were averaged and confirmed, and the finally aggregated result was t for the value of the negative control group that induced only stress. -test was conducted and the statistical significance was * p <0.05.

Experimental group Medicinal substances
Treatment concentration Number of stress granules per cell (mean±sem) IPTG (no treatment group) 0μM 2.30±0.17 IPTG+NaAsO2 (negative control) 0μM 6.14±0.66 IPTG+NaAsO2+Arimoclomol (positive control) 10 μM 3.68±0.32 ^* IPTG+NaAsO2+Epiandrosterone 1 μM 4.13±0.30 ^* IPTG+NaAsO2+Topiramate 1 μM 4.03±0.29 ^*

Experimental group
Medicinal substances
Treatment concentration Granule-free cells/total cells ratio (mean±sem) IPTG (no treatment group) 0μM 0.34±0.029 IPTG+NaAsO2 (negative control) 0μM 0.15±0.026 IPTG+NaAsO2+Arimoclomol (positive control) 10 μM 0.25±0.030 ^* IPTG+NaAsO2+Epiandrosterone 1 μM 0.24±0.031 ^* IPTG+NaAsO2+Topiramate 1 μM 0.32±0.034 ^*

Looking at the results of Table 1 and Figure 1, the experimental group treated with 1 μM epiandrosterone and 1 μM topiramate and the 10 μM arimoclomol treated group as a positive control showed a statistically significant reduction in stress granule compared to the negative control treated with only sodium arsenite. In the negative control group, when about 6 stress granules were formed per cell, the effects of 1μM epiandrosterone, 1μM topiramate, and 10μM arimoclomol were similar at about 3.5 levels per cell, showing a statistically insignificant difference, but positive in terms of concentration. Similar results were obtained through epiandrosterone and topiramate at concentrations 10 times less than that of arimoclomol, the control group, so it can be confirmed that the lowering effect of the stress granule was remarkable compared to the positive control.

Apart from the direct coefficient of stress granule, the ratio of cells in which stress granule was not generated in Table 2 and FIG. 2, which is a result of measuring the ratio to the total number of cells by counting granule free with respect to the occurrence of stress granule, is also Table 1 And conclusions similar to the results of FIG. 1 were drawn.

Therefore, it was confirmed that when epiandrosterone and topiramate were treated by applying oxidative stress to cells, the aggregation of stress granules bound to ATXN2 by overexpression of TDP-43 protein was effectively reduced. As a result, epiandrosterone and topiramate were found to be in the central nervous system ( Central nervous system, CNS) diseases, such as amyotrophic lateral sclerosis or frontal dementia.

Claims (3)

Central arising from the generation of stress granules in cells by inhibiting abnormal aggregation of TDP-43 by inhibiting ATXN2 containing any one selected from the group consisting of epiandrosterone and its pharmaceutically acceptable salts as an active ingredient. A pharmaceutical composition for the prevention and treatment of nervous system diseases.
According to claim 1, wherein the central nervous system disease is amyotrophic lateral sclerosis or frontal dementia.
The frontal lobe caused by the generation of stress granules in cells by inhibiting abnormal aggregation of TDP-43 by inhibiting ATXN2 containing any one selected from the group consisting of Topiramate and its pharmaceutically acceptable salts as an active ingredient. Pharmaceutical composition for the prevention and treatment of sexual dementia.

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