KR20240120380A - Novel composition for inhibiting histone deacetylase - Google Patents

Abstract

The present invention relates to a novel composition for inhibiting histone deacetylase (HDAC). Silver sulfadiazine of the present invention has excellent ability to inhibit histone deacetylase (HDAC) activity. Therefore, it can be used as an HDAC inhibitor, and by inhibiting HDAC activity, it can be used to treat various related diseases (for example, metabolic diseases such as obesity and diabetes).

Description

Novel composition for inhibiting histone deacetylase {Novel composition for inhibiting histone deacetylase}

The present invention relates to a novel composition for inhibiting histone deacetylase (HDAC), and more specifically, to a composition for inhibiting HDAC containing silver sulfadiazine.

Histone deacetylase (HDAC) is involved in the epigenetic regulation of genes, is associated with various metabolic syndromes such as diabetes and obesity, and has been reported to have anti-cancer effects. Since HDAC1 was named, a total of 18 subtypes of HDAC have been discovered. Trichostatin A (TSA) is the most widely used HDAC inhibitor, and was originally known as a parasite drug, but was later found to be effective as an HDAC inhibitor. HDAC inhibitors are highly likely to be used as new treatments for various diseases, and ongoing research is underway.

Meanwhile, Silver [(4-aminophenyl)sulfonyl](pyrimidin-2-yl)azanide, also known as silver sulfadiazine, is known as an antibiotic that inhibits bacterial growth, but has been reported in relation to HDAC inhibitors. There is no

As a result of diligent efforts to develop a new HDAC inhibitor, the present inventors completed the present invention by confirming that silver sulfadiazine has an excellent inhibitory effect on HDAC 4, HDAC 5, and HDAC 11 activities.

The present invention provides a novel composition for inhibiting histone deacetylase (HDAC).

In order to achieve the above object, the present invention provides a composition for inhibiting histone deacetylase (HDAC) containing silver sulfadiazine.

In the present invention, the histone deacetylase (HDAC) is characterized in that it is at least one selected from the group consisting of HDAC 4, HDAC 5, and HDAC 11.

In the present invention, the histone deacetylase (HDAC) is HDAC 4, and the composition may be characterized as containing 0.001 μM or more of silver sulfadiazine.

In the present invention, the histone deacetylase (HDAC) is HDAC 5, and the composition may be characterized as containing 0.001 μM or more of silver sulfadiazine.

In the present invention, the histone deacetylase (HDAC) is HDAC 11, and the composition may be characterized as containing 0.1 μM or more of silver sulfadiazine.

Additionally, the present invention provides a composition for preventing or treating metabolic diseases containing silver sulfadiazine as an active ingredient.

In the present invention, the composition may be characterized as preventing or treating metabolic diseases by inhibiting HDAC activity, and the target diseases may include obesity, diabetes, etc.

Silver sulfadiazine of the present invention has excellent ability to inhibit histone deacetylase (HDAC) activity, so it can be used as an HDAC inhibitor. By inhibiting the HDAC activity, it can be used to treat various related diseases (e.g., obesity). It can be used to treat metabolic diseases such as diabetes, etc.

Figure 1 relates to the results of analyzing the ability of silver sulfadiazine to inhibit HDAC4 activity. The ability to inhibit HDAC4 activity was compared using TSA as a positive control and FT895 as a negative control.
Figure 2 relates to the results of analyzing the ability of Silver Sulfadiazine to inhibit HDAC5 activity. The ability to inhibit HDAC5 activity was compared using TSA as a positive control and FT895 as a negative control.
Figure 3 relates to the results of analyzing the ability of Silver Sulfadiazine to inhibit HDAC11 activity. TSA and FT895 were used as positive controls to compare their ability to inhibit HDAC11 activity.

Hereinafter, the present invention will be described in detail.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a composition for inhibiting HDAC containing silver sulfadiazine.

In one embodiment of the present invention, the histone deacetylase (HDAC) may be characterized as one or more selected from the group consisting of HDAC 4, HDAC 5, and HDAC 11, but is not limited thereto.

In another embodiment of the present invention, the silver sulfadiazine may be included in an amount of 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, but is not limited thereto. However, as a result of the analysis of the activity inhibition effect on HDAC4 in the present invention, the IC50 of silver sulfadiazine was measured to be 0.1727μM, and when silver sulfadiazine is included at less than 0.001μM, it effectively inhibits Histone Deacetylase 4 (HDAC4). Problems that cannot be suppressed may arise.

In another embodiment of the present invention, the silver sulfadiazine may be contained in an amount of 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, and is limited thereto. However, as a result of the analysis of the activity inhibition effect on HDAC5 in the present invention, the IC50 of silver sulfadiazine was measured to be 0.1203 μM, and when silver sulfadiazine is included at less than 0.001 μM, histone deacetylase 5 (HDAC5) A problem may arise in that it cannot be effectively suppressed.

In another embodiment of the present invention, the silver sulfadiazine may be contained in an amount of 0.1 μM or more, preferably 1.0 μM or more, more preferably 10.0 μM or more, and is limited thereto. However, as a result of the analysis of the activity inhibition effect on HDAC11 in the present invention, the IC50 of silver sulfadiazine was measured to be 26.42μM, and when silver sulfadiazine is included at less than 0.001μM, histone deacetylase 11 (HDAC11) A problem may arise in that it cannot be suppressed effectively.

From another perspective, the present invention relates to a composition for preventing or treating metabolic diseases, comprising silver sulfadiazine as an active ingredient.

In one embodiment of the present invention, the composition may prevent or treat metabolic diseases by inhibiting HDAC activity, and the target diseases may include obesity, diabetes, etc.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

[Example]

Analysis of HDAC4 activity inhibition ability

(Example 1-1) Experimental materials

Fluorogenic HDAC4 Assay Kit to analyze HDAC4 activity inhibition ability (BPS Bioscience/Dongin Biotech), HDAC4 human recombinant enzyme (BPS Bioscience/Dongin Biotech), Fluorogenic HDAC substrate class 2A (BPS Bioscience/Dongin Biotech), 2X HDAC assay developer (BPS Bioscience/Dongin Biotech), Trichostatin A (BPS Bioscience) /Dongin Biotech), HDAC Assay Buffer (BPS Bioscience/Dongin Biotech), Bovine serum albumin (RDTech/Dongin Biotech), black microtiter plate (NUNC/Dongin Biotech), and Spectramax iD3 (Molecular Device/Power Lab) were used.

(Example 1-2) Method for measuring HDAC4 activity

Dilute the sample, silver sulfadiazine, in HDAC assay buffer to prepare concentrations of 100μM, 10μM, 1μM, 0.1μM, 0.01μM, and 0.001μM. Trichostatin A and FT895, which are control substances, are similarly diluted in HDAC assay buffer and prepared to have concentrations of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM. Fluorogenic HDAC substrate class 2A is diluted in HDAC assay buffer to prepare 20μM, and bovine serum albumin is dissolved in water to prepare at a concentration of 0.1%. Then, mix the prepared HDAC substrate and bovine serum albumin with HDAC assay buffer to create a master mixture. Dispense 40 μl of the prepared master mixture into each well of the black microtiter plate. Add 5 μl each of the prepared sample and control material to the well containing the master mixture, then add 5 μl each of HDAC4 human recombinant enzyme and react at 37°C for 30 minutes. After 30 minutes, add 50 μl of 2X HDAC assay developer to each well and react at room temperature for 15 minutes. After 15 minutes, read the plate using spectramax iD3 at Excitation 360nm and Detection 460nm. Subtract the blank value from all measured values and then draw a log graph to check the results.

(Example 1-3) HDAC4 activity inhibition ability analysis results

To determine whether silver sulfadiazine has an activity-inhibiting effect on HDAC4, the ability to inhibit HDAC4 activity was measured using silver sulfadiazine and a drug known as an HDAC inhibitor. Silver sulfadiazine was used as an experimental group, Trichostatin A (TSA), known as a Pan HDAC inhibitor, was used as a positive control group, and FT895, known to have a specific activity inhibition effect on HDAC 11, was used as a negative control group. In addition, the activity inhibition efficacy of each substance was measured using the HDAC4 activity fluorescence analysis kit, and the IC50, which is the concentration showing 50% activity inhibition, was calculated and compared.

As a result, as shown in Figure 1 and Table 1 below, the IC50 of silver sulfadiazine, the experimental group, was measured at 0.1727 μM, and TSA, the positive control group, was measured at 13.62 μM. In the case of FT895, a negative control group, it did not show the ability to inhibit HDAC4 activity. Through this, it was confirmed that silver sulfadiazine has an excellent ability to inhibit HDAC4 activity.

Analysis of HDAC5 activity inhibition ability

(Example 2-1) Experimental materials

Fluorogenic HDAC5 Assay Kit to analyze HDAC5 activity inhibition ability (BPS Bioscience/Dongin Biotech), HDAC5 human recombinant enzyme (BPS Bioscience/Dongin Biotech), Fluorogenic HDAC substrate class 2A (BPS Bioscience/Dongin Biotech), 2X HDAC assay developer (BPS Bioscience/Dongin Biotech), Trichostatin A (BPS Bioscience) /Dongin Biotech), HDAC Assay Buffer (BPS Bioscience/Dongin Biotech), Bovine serum albumin (RDTech/Dongin Biotech), black microtiter plate (NUNC/Dongin Biotech), and Spectramax iD3 (Molecular Device/Power Lab) were used.

(Example 2-2) Method for measuring HDAC5 activity

Dilute the sample, silver sulfadiazine, in HDAC assay buffer to prepare concentrations of 100μM, 10μM, 1μM, 0.1μM, 0.01μM, and 0.001μM. Trichostatin A and FT895, which are control substances, are similarly diluted in HDAC assay buffer and prepared to have concentrations of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM. Fluorogenic HDAC substrate class 2A is diluted in HDAC assay buffer to prepare 20μM, and bovine serum albumin is dissolved in water to prepare at a concentration of 0.1%. Then, mix the prepared HDAC substrate and bovine serum albumin with HDAC assay buffer to create a master mixture. Dispense 40 μl of the prepared master mixture into each well of the black microtiter plate. Add 5 μl each of the prepared sample and control material to the well containing the master mixture, then add 5 μl each of HDAC5 human recombinant enzyme and react at 37°C for 30 minutes. After 30 minutes, add 50 μl of 2X HDAC assay developer to each well and react at room temperature for 15 minutes. After 15 minutes, read the plate using spectramax iD3 at Excitation 360nm and Detection 460nm. Subtract the blank value from all measured values and then draw a log graph to check the results.

(Example 2-3) HDAC5 activity inhibition ability analysis results

To determine whether silver sulfadiazine has an activity-inhibiting effect on HDAC5, the ability to inhibit HDAC5 activity was measured using silver sulfadiazine and a drug known as an HDAC inhibitor. Silver sulfadiazine was used as an experimental group, Trichostatin A (TSA), known as a Pan HDAC inhibitor, was used as a positive control group, and FT895, known to have a specific activity inhibition effect on HDAC 11, was used as a negative control group. In addition, the activity inhibition efficacy of each substance was measured using the HDAC5 activity fluorescence analysis kit, and the IC50, which is the concentration showing 50% activity inhibition, was calculated and compared.

As a result, as shown in Figure 2 and Table 1 below, the IC50 of silver sulfadiazine, the experimental group, was measured at 0.1203 μM, and TSA, the positive control group, was measured at 13.05 μM. In the case of FT895, a negative control group, it did not show the ability to inhibit HDAC5 activity. Through this, it was confirmed that silver sulfadiazine has an excellent ability to inhibit HDAC5 activity.

Analysis of HDAC11 activity inhibition ability

(Example 3-1) Experimental materials

Fluorogenic HDAC11 Assay Kit to analyze HDAC11 activity inhibition ability (BPS Bioscience/Dongin Biotech), HDAC11 human recombinant enzyme (BPS Bioscience/Dongin Biotech), Fluorogenic HDAC substrate class 4 (BPS Bioscience/Dongin Biotech), 2X HDAC assay developer (BPS Bioscience/Dongin Biotech), Trichostatin A (BPS Bioscience) /Dongin Biotech), HDAC Assay Buffer (BPS Bioscience/Dongin Biotech), Bovine serum albumin (RDTech/Dongin Biotech), black microtiter plate (NUNC/Dongin Biotech), and Spectramax iD3 (Molecular Device/Power Lab) were used.

(Example 3-2) Method for measuring HDAC11 activity

Dilute the sample, silver sulfadiazine, in HDAC assay buffer to prepare concentrations of 100μM, 10μM, 1μM, 0.1μM, 0.01μM, and 0.001μM. Trichostatin A and FT895, which are control substances, are similarly diluted in HDAC assay buffer and prepared to have concentrations of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM. Fluorogenic HDAC substrate class 4 is diluted in HDAC assay buffer to prepare 20μM, and bovine serum albumin is dissolved in water to prepare at a concentration of 0.1%. Then, mix the prepared HDAC substrate and bovine serum albumin with HDAC assay buffer to create a master mixture. Dispense 40 μl of the prepared master mixture into each well of the black microtiter plate. Add 5 μl each of the prepared sample and control material to the well containing the master mixture, then add 5 μl each of HDAC11 human recombinant enzyme and react at 37°C for 30 minutes. After 30 minutes, add 50 μl of 2X HDAC assay developer to each well and react at room temperature for 15 minutes. After 15 minutes, read the plate using spectramax iD3 at Excitation 360nm and Detection 460nm. Subtract the blank value from all measured values and then draw a log graph to check the results.

(Example 3-3) HDAC11 activity inhibition ability analysis results

To determine whether silver sulfadiazine has an activity-inhibiting effect on HDAC11, the ability to inhibit HDAC11 activity was measured using silver sulfadiazine and a drug known as an HDAC inhibitor. Silver sulfadiazine was used as an experimental group, and Trichostatin A (TSA), known as a Pan HDAC inhibitor, and FT895, known to have a specific inhibitory effect on HDAC 11, were used as positive controls. In addition, the activity inhibition efficacy of each substance was measured using the HDAC11 activity fluorescence analysis kit, and the IC50, which is the concentration showing 50% activity inhibition, was calculated and compared.

As a result, as shown in Figure 3 and Table 1 below, the IC50 of silver sulfadiazine, an experimental group, was measured at 26.42 μM, TSA, one of the positive control groups, was measured at 26.31 μM, and the remaining one, FT895, was measured at 0.3878 μM. Through this, it was confirmed that silver sulfadiazine has an excellent ability to inhibit HDAC11 activity.

Table 1 above summarizes the results of analysis of the activity inhibition ability of Silver Sulfadiazine, TSA, and FT895 against HDAC4, 5, and 11. In other words, as a result of comparing the IC50 values for HDAC4, 5, and 11 of Silver Sulfadiazine and TSA and FT895, silver sulfadiazine has excellent inhibition ability of HDAC 4, 5, and 11, especially HDAC4 and 5 inhibition ability. It was confirmed to be very excellent.

Claims (7)

A composition for inhibiting histone deacetylase (HDAC) containing silver sulfadiazine as an active ingredient.
According to paragraph 1,
The composition, wherein the histone deacetylase (HDAC) is at least one selected from the group consisting of HDAC 4, HDAC 5, and HDAC 11.
According to paragraph 1,
The histone deacetylase (HDAC) is HDAC 4, and the composition contains 0.001 μM or more of silver sulfadiazine.
According to paragraph 1,
The histone deacetylase (HDAC) is HDAC 5, and the composition contains 0.001 μM or more of silver sulfadiazine.
According to paragraph 1,
The histone deacetylase (HDAC) is HDAC 11, and the composition contains 0.1 μM or more of silver sulfadiazine.
A composition for preventing or treating metabolic diseases containing silver sulfadiazine as an active ingredient.
According to clause 6,
The composition is a preventive or therapeutic composition, characterized in that it prevents or treats metabolic diseases by inhibiting HDAC activity.