KR20220112318A - Pharmaceutical composition for preventing or treating ciliopathy comprising metabolites - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating ciliary abnormalities containing at least one from a group consisting of acetyl-CoA, octanoyl-CoA and acetyl-CoA precursor as an active ingredient. More specifically, as the reduction of acetyl-CoA, the length of primary cilia, and the number of ciliated cells in cells in which the expression of the HSD17B4 gene, which regulates the β-oxidation activity of peroxisomes, is inhibited, is confirmed and as an increase in the length of primary cilia and the number of ciliated cells, which was decreased, is confirmed in the HSD17B4 expression-suppressing cells treated with acetyl-CoA or octanoyl-CoA, the composition consisting of the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) or acetyl-CoA precursor can be used as a pharmaceutical composition and health food for preventing or treating ciliary abnormalities caused by primary ciliary abnormalities.

Description

A pharmaceutical composition for preventing or treating ciliary disease, comprising metabolites as an active ingredient;

The present invention contains one or more of the group consisting of acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors as an active ingredient, and treats ciliary disorders induced by primary ciliary abnormalities It relates to a composition for

Primary cilia are organelles protruding from the cell surface, and are composed of microtubules, which are central skeletons, and a specialized plasma membrane. Almost all eukaryotic cells are immobile. Has one primary cilia. In the past, primary cilia were considered to be a product of evolutionary degeneration with no special function, but through recent studies, primary cilia serve as the antenna of the cell for external stimuli or various sensory signals (visual, olfactory, auditory, kinesthetic, osmotic, etc.) As it is known that Hedgehog, TGF-b, PDGF, AMPK, mTOR, and autophagy, which are crucial intracellular signaling processes, occur through primary cilia, the importance of primary cilia as a “signaling hub” is emerging. .

Ciliary disorders include polycystic kidney diseas (PKD), infertility, developmental disorders, situs inversus, as well as metabolic diseases such as obesity, diabetes, It appears in various forms, such as cancer. In particular, among ciliary disorders-related diseases, neurodegenerative disease, hearing loss, sensory dysfunction such as retinal degeneration, kidney disease, metabolic disease ( Metabolic disorders) are diseases associated with dysfunction of the endoplasmic reticulum (ER), mitochondria, and peroxisome, which are important organelles in cells, and these organelles are responsible for intracellular energy metabolism ) and plays an important role in enzymatic activity.

As such, it has been reported that primary cilia are involved in the onset of various diseases, but an effective therapeutic agent for ciliary abnormalities to fundamentally prevent such onset has not yet been reported.

Republic of Korea Patent Publication No. 10-2017-0141434 (published on December 26, 2017)

The present invention provides a composition containing acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) or an acetyl-CoA precursor as an active ingredient for the prevention or treatment of ciliary disorders induced by primary ciliary abnormalities would like to provide

The present invention provides a pharmaceutical composition for preventing or treating ciliary disease, comprising at least one active ingredient from the group consisting of acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors. .

The present invention provides a health food for preventing or improving ciliary disease disease containing at least one active ingredient from the group consisting of acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors. .

In addition, the present invention comprises the steps of treating a candidate substance to a cell line in which expression of a β-oxidation activity regulatory gene is suppressed;

identifying one or more levels of increase in the group consisting of the length of primary cilia and the number of ciliated cells in the cell line treated with the candidate substance; and

It provides a screening method for a therapeutic agent for ciliary disorders, comprising comparing the increase in the length of the primary cilia and the number of ciliated cells with a normal control group.

According to the present invention, a decrease in acetyl-CoA and a decrease in the length of primary cilia and the number of ciliated cells were confirmed in cells in which the expression of the HSD17B4 gene, which regulates the β-oxidation activity of peroxisomes, was suppressed. On the other hand, as it was confirmed that the length of primary cilia and the number of ciliated cells, which were decreased in HSD17B4 expression-suppressed cells treated with acetyl-CoA or octanoyl-CoA, were confirmed, the acetyl-CoA, octanoyl-CoA or acetyl-CoA precursors A composition containing as an active ingredient may be provided as a pharmaceutical composition and health food for preventing or treating ciliary disease caused by primary cilia abnormality.

1 is a result confirming the effect of increasing ciliary cells according to acetyl-CoA, octanoyl-CoA, and acetyl-CoA precursor (acetate/Acetate) treatment.
2 is a result confirming ciliation inhibition and acetyl-CoA level change in cells knocked out HSD17B4, a PDB-related gene.
3 is a result confirming ciliation inhibition and acetyl-CoA level change in patient-derived cells having an HSD17B4 mutant gene.
Figure 4 is a result confirming the increase in ciliation and acetyl-CoA level change according to acetyl-CoA and octanoyl-CoA treatment.
5 is a result confirming the ciliary abnormality and acetyl-CoA recovery effect according to acetyl-CoA and octanoyl-CoA treatment in PDB patient-derived fibroblasts.
6 is a result confirming the effect of restoring intracellular cilia-associated signaling according to acetyl-CoA treatment in HSD17B4 knockout cells.
7 is a result confirming the recovery of neuronal apoptosis by acetyl-CoA and octanoyl-CoA treatment for neuronal cell death according to MPP ⁺ treatment, a mitochondrial toxin.
8 is a result confirming the cell survival effect of acetyl-CoA and octanoyl-CoA treatment on neuronal cell death according to inhibition of primary ciliary activity.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating ciliary disease disease containing at least one from the group consisting of acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors as an active ingredient. can

The acetyl-CoA precursor may be selected from the group consisting of acetate, pyruvate, and citrate.

The ciliary disease may be selected from the group consisting of peroxisome biogenesis disorders, neurodegenerative diseases, cancer diseases, and obesity caused by primary ciliary abnormalities.

More specifically, the ciliary disease is polycystic kidney disease (PKD), infertility, developmental disorder, microcephaly, facial dysmorphism, polydactyly, To be selected from the group consisting of situs inversus, cognitive defect, loss of hearing and vision, retinopathy, hypotension, fibrosis, etc. However, the present invention is not limited thereto.

The peroxisome biogenesis disorders include Zellweger syndrome (ZSD), rhizomelic chondrodysplasia punctata/RCDP, β-oxidation enzyme defects, Refsum's disease ( Infantile Refsum Disease) and neonatal adrenoleukodystrophy (NALD) may be selected from the group consisting of, but is not limited thereto.

The degenerative neurological disease may be one selected from the group consisting of Alzheimer's disease, dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS) and memory loss. , but is not limited thereto.

The cancer disease may be selected from the group consisting of chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia and acute lymphocytic leukemia, but is not limited thereto.

The acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors may increase the length of primary cilia and the number of ciliated cells.

In addition, the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors increase phosphorylation of AKT and SMAD2/3 in HSD17B4 expression-suppressed cells to restore ciliary-associated signaling. can

According to an embodiment of the present invention, the SH-SY5Y human neuroblastoma cell line (HSD17B4 knockout) in which HSD17B4, a gene related to peroxisome biogenesis disorders (PBD) that regulates β-oxidation activity of peroxisome, is knocked out Immunofluorescence staining using ARL13b antibody and hoechst dye to identify ciliated cells in the wild type (SH-SY5Y/Cas9 cell line (WT)) with normal expression of SH-SY5Y/Cas9 HSD17B4 KO (KO)) and HSD17B4 gene. As a result of confirming the ciliary morphology of the cells through a fluorescence microscope after performing , it was confirmed that the number of cells having primary cilia and the length of primary cilia decreased in HSD17B4 gene KO cells as shown in FIG. 2 . In addition, as a result of confirming the amount of intracellular acetyl-CoA, it was confirmed that the amount of acetyl-CoA in KO cells was significantly reduced by about 10% or more compared to normal-expressing neuroblasts.

On the other hand, according to another embodiment, acetyl-CoA (100 μM, Sigma) and octanoyl-CoA (100 μM , Sigma) after 24 hours of treatment, the cells were washed with normal Dulbeccoic acid buffered saline (DPBS) and immunotransfection and intracellular acetyl CoA quantitative analysis were performed. The number of cilia-bearing cells and the length of primary cilia were restored to more than normal levels by acetyl-CoA treatment, and the amount of acetyl-CoA, which was reduced by HSD17B4 knockout, was restored by acetyl-CoA and octanoyl-CoA treatment. was able to confirm that

From the above results, it was confirmed that acetyl-CoA and octanoyl-CoA exhibit an effect of restoring the decrease in the length of primary cilia and the number of ciliated cells induced by peroxisome dysfunction or decrease in acetyl-CoA.

In one embodiment of the present invention, the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursor containing at least one from the group consisting of an active ingredient for preventing or treating ciliary disease For the pharmaceutical composition, any one formulation selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids may be used according to a conventional method.

In another embodiment of the present invention, suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, It may further include one or more additives selected from the group consisting of surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

Preferred dosages of the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) or acetyl-CoA precursor may vary depending on the subject's condition and weight, the type and degree of disease, drug form, administration route and duration. may vary and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

The present invention provides a health food for preventing or improving ciliary disease disease containing at least one from the group consisting of acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors as an active ingredient. can

The health food is used together with other foods or food additives in addition to the acetyl-CoA, octanoyl-CoA and acetyl-CoA precursors, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the intended use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but in the case of long-term intake for health and hygiene or health control, it may be less than or equal to the above range, It is clear that the ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

The type of health food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

In addition, the present invention comprises the steps of treating a candidate substance to a cell line in which expression of a β-oxidation activity regulatory gene is suppressed; Identifying one or more levels of increase in the group consisting of the length of primary cilia and the number of ciliated cells in the cell line treated with the candidate substance; And it may provide a method for screening a therapeutic agent for ciliary abnormalities comprising the step of comparing the increase in the length of the primary cilia and the number of ciliated cells with a normal control group.

The cell line in which the expression of the β-oxidation activity regulatory gene is suppressed may be one in which the primary cilia length and the number of ciliated cells are reduced.

The β-oxidation activity regulatory gene is HSD17B4 (NM_000414), ATP Binding Cassette Subfamily D Member 1 (ABCD1) (NM_000033), Acyl-CoA Oxidase 1 (ACOX1) (NM_001185039), Solute Carrier Family 25 Member 20 (CACT) (NM_000387) and Trypsin Domain Containing 1 (TYSND1) (NM_173555) may be selected from the group consisting of.

Hereinafter, in order to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Preparation of human retinal pigment epithelial cells and neuroblastoma cell lines

Human telomerase-immortalized retinal pigmented epithelial cells (htRPE cells) and neuroblastoma (SH-SY5Y) were purchased from ATCC (American Type Culture Collection), 5% (v/v) CO ₂ in an incubator, 37 It was cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen, CA, USA) at a temperature of ℃.

2. Construction of HSD17B4 knock-out cell line using CRISPR/Cas9 system

The cultured neuroblastoma (SH-SY5Y) was transfected with the vector hCMV-PuroR-Cas9 Expression Plasmid DNA expressing the Cas9 protein purchased from Dharmacon (Lafayette, CO, USA) to transfect a Cas9-expressing cell line (SY5Y/Cas9). ) was produced.

As a specific method, after culturing 4×10 ⁵ neuroblastoma cells in a 100 mm cell culture dish for 24 hours, add 5 μg of Cas9 plasmid to 500 μl Opti-MEM (GIBCO), mix well, and mix well with 500 μl Opti- After mixing 10 μl of Lipofectamine ^® 2000 with MEM, mix the above two mixtures well and react at room temperature for 20 minutes, add 4 ml of Opti-MEM, and treat the cells with 5 ml of final mixture in a dish vector was injected. After 24 hours, the cells were treated with the antibiotic puromycin at a concentration of 12 μg/ml and selected for 7 days. Thereafter, the selected cells were cultured by putting only single cells per well in a 96-well plate (single cell dropping), and then colonies were selected and cultured. In the selected colony cells, expression of Cas9 protein was confirmed by Western blot. For the production of the SY5Y/Cas9 cell line, an HSD17B4 knock-out cell line (SY5Y/HSD17B4 KO), a crRNA targeting the HSD17B4 gene (Edit-R Human HSD17B4 (3295) crRNA) and a guide RNA 150 pmol of tracrRNA for production (Edit-R CRISPR-Cas9 Synthetic tracrRNA) was mixed with 500 μl of Opti-MEM in a 1:1 ratio of 300 pmol each, and 10 μl of Lipofectamine® 2000 was mixed with 500 μl of Opti-MEM. After the mixture was reacted at room temperature for 20 minutes, the vector was injected into the SY5Y/Cas9 cell line prepared by adding 4 ml of Opti-MEM. After 48 hours, colonies were selectively cultured by culturing only single cells in a 96-well plate, and then, it was confirmed whether HSD17B4 expression was knocked out by Western blot. Thereafter, the prepared SY5Y/Cas9 WT and HSD17B4 KO cell lines were in a 5% (v/v) CO ₂ incubator, under a temperature condition of 37° C., with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin. - Incubated in DMEM supplemented with streptomycin (Invitrogen).

3. Preparation of HSD17B4 Mutant Patient-derived Cell Lines

A normal human skin-derived fibroblast line (GM00969) and a human HSD17B4 gene mutated (422Del2, D-Bifunctional Protein Deficiency) patient skin-derived fibroblast cell line (GM13263) were purchased from Coriell Institute (Camden, NJ, USA). In a 5% (v/v) CO ₂ incubator, it was cultured in DMEM supplemented with 15% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin at a temperature of 37°C.

4. Acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetate (Acetate) treatment

In order to confirm the change in primary cilia formation by acetyl-CoA, octanoyl-CoA and acetate (Sigma-Aldrich, St. Louis, MO) in the cell line, cells were cultured in a 12-well culture plate for 24 hours. , acetyl-CoA, octanoyl-CoA and acetate were each included in the medium at a concentration of 100 μM and treated for 24 hours.

5. Immunofluorescence staining using a fluorescence microscope

After culturing the cells in a 12-well culture plate (SPL Life Sciences, South Korea) at a cell count of 8 × 10 ⁴ per well for 24 hours, from the time when the cells reached about 90%, a control or test substance containing only the medium After culturing for 24 hours in a medium containing 1 μM of SAG, 100 μM of Acetyl-CoA and Octanoyl-CoA, respectively, cells were fixed with 4% (w/v) paraformaldehyde and primary cilia were stained. Primary cilia staining was specifically performed with anti-ARL13B antibody (1/1000 dilution, Proteintech, diluted in PBS containing 1% (v/v) BSA and 0.1% (v/v) triton™ X-100 (PBS-T)). green) and incubated overnight at 4°C. After that, Alexa Fluor 488 goat anti-rabbit (Invitorgen) was diluted 1/1000 with the PBS as a secondary antibody and incubated for 2 hours at room temperature. -Fisher, blue) was used for 10 minutes at room temperature, and IX71 (IX71, Olympus, Japan) was used for a fluorescence microscope.

6. Primary cilia formed cells and primary cilia length measurement

Cells with primary cilia were imaged on a fluorescence microscope (IX71, Olympus, Japan) using Cell-Sense Standard software (>100 cells per experiments, n=3), and elongation compared to the total number of cells on the captured image. The number of cells with elongated cilia was measured and used as the value of ciliated cells (%). The length of the primary cilia was determined by measuring the length of each primary cilia on the captured image using the Free-hand Line Selection Tool (Olympus EuropaHolding GmbH, Hamburg, Germany) of Cell-Sense Standard software, and then measuring the total length of the primary cilia. The average value of was used. Each analyzed value was graphed using GraphPad Prism 8 (GraphPad Software, San Diego, CA).

7. Check the amount of acetyl-CoA in the cell

HSD17B4 WT and KO neuroblastoma and fibroblasts were treated with acetyl-CoA or octanoyl-CoA at 100 μM each for 24 hours to collect only cell pellets, and then use Acetyl-Coenzyme A assay kit (Sigma-Aldrich) Thus, according to the manufacturer's instructions, the amount of Acetyl-CoA in the cell was confirmed by measuring the degree of fluorescence expression at a wavelength of 535/587 nm.

8. siRNA transfection and reagent processing

IFT88 siRNA targeting IFT88 (5'-CCGAAGCACUUAACACUUA-3') and negative control siRNA (scramble siRNA, Sc) (5'-CCUACGCCACCAAUUUCGUUU-3') were purchased from Genolution. Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the neuroblastoma cultured above with siRNA. Specifically, put 2 μl (200 pmol) of each siRNA in 50 μl Opti-MEM (GIBCO) per well, mix well, and mix 1.6 μl of Lipofectamine® 2000 in 50 μl Opti-MEM. After mixing the mixture well and reacting at room temperature for 20 minutes, 400 μl of Opti-MEM was further added, and the cells were treated with 500 μl of the final mixture in the well for transfection. After reacting at 37° C. for about 2 hours, it was replaced with a new DMEM medium and cultured. After 48 hours, the transfected neuroblastoma was additionally treated with MPP+ 2.5 mM, a mitochondrial respiratory chain inhibitor, for 24 hours, and then harvested the cells and changed apoptosis through western blotting. to measure the change in cell viability through CCK-8 assay.

9. Western blotting analysis

All samples prepared from the cells were prepared in 2× Laemmli sample buffer (2Х Laemmli sample buffer, 62.5 mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] SDS, 5% [v] /v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue) (Bio-Rad, Hercules, CA, USA) was used. All cellular proteins were quantitatively measured using Bradford solution (Bio-Rad). The samples were then separated on SDS-PAGE gels, transferred to PVDF membrane (Bio-Rad), and 4% (25 mM Tris, 140 mM NaCl, and 0.05% [v/v] Tween® 20) in TBST ( w/v) After blocking with skim milk, the membrane was reacted with primary antibodies at 4° C. overnight. Anti-Cas9 antibody (anti-Cas9 antibody, 1:2000) was purchased from Novus biologicals (Littleton, CO), and anti-HSD17B4 antibody (anti-HSD17B4 antibody, 1:1000) was from Abcam (Cambridge, UK), anti -p-AKT (anti-p-AKT antibody, 1:3000), anti-p-SMAD2/3 (anti-p-SMAD2/3 antibody, 1:1000) and anti-cleaved caspase-3 (anti-cleaved caspase) -3 antibody, 1:3000) was purchased from Cell Signaling Technology (Danvers, MA), and anti-actin antibody (anti-Actin antibody, 1:10000) was purchased from Millipore (Temecula, CA). For protein detection, the membrane was reacted for 2 hours at room temperature using an HRP-conjugated secondary antibody (horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockford, IL)), and the luminescence signal was obtained from Clarity Western ECL substrate. (ATTO, Moto Asakusa, Tokyo, Japan) The expression level of each protein was confirmed using the AE-9300 Ez-Capture MG Hours Image Saver HR image capture tool (WSE-7120L, ATTO, Tokyo, Japan) did.

10. Check cell viability

After 48 hours of transfection of neuroblastoma with IFT88 siRNA, cells treated with MPP+ for 24 hours were treated with CCK-8 (Cell Counting Kit-8, Doindo Laboratories, Kumamoto, Japan) at 450 nm wavelength according to the manufacturer's instructions. The degree of cell viability was analyzed by measuring the absorbance.

<Example 1> Acetyl-CoA (Acetyl-CoA) and octanoyl-CoA (Octanoyl-CoA) according to the increase in ciliated cells confirmed

Acetyl-CoA (100 μM), octanoyl-CoA (100 μM), acetyl-CoA precursors in human retinal epithelial cells (RPE cells) After 24 hours of treatment with phosphorus acetate (Acetate, 10 mM) and positive control SAG (Smoothend Agonist, 1 μM), immunofluorescence staining (green) and nuclear staining using ARL13b protein antibody and Hoechst dye used as a staining marker for cilia After (blue), the length of the primary cilia of the stained cells and the ciliated cells were analyzed using a fluorescence microscope.

As a result, as shown in FIG. 1, in human retinal epithelial cells (RPE cells) treated with acetyl-CoA, octanoyl-CoA and acetyl-CoA precursors (Acetate), ARL13b protein antibody used as a staining marker for cilia and hoechst dye were used to immunize Staining (green) and nuclear staining (blue) were confirmed. As a result of analyzing the length of primary cilia and ciliated cells of the stained cells through a fluorescence microscope, acetyl-CoA, octanoyl CoA and acetyl-CoA It was confirmed that the length of primary cilia and the number of ciliated cells were increased by the precursor (Acetate) treatment.

<Example 2> Confirmation of ciliation and acetyl-CoA levels in HSD17B4 knockout cells

A cell line in which HSD17B4, a gene related to peroxisome biogenesis disorders (PBD) that regulates β-oxidation activity, was knocked out was prepared in the SH-SY5Y cell line using the gene scissors method (SH-SY5Y/Cas9 cell line (WT)) and HSD17B4 knockout SH-SY5Y/Cas9 HSD17B4 KO (KO)). Immunofluorescence using ARL13b antibody and hoechst dye to identify ciliated cells in wild-type (WT) with normal expression of HSD17B4 gene and knockout (KO) human neuroblastoma cells with suppressed gene expression (SH-SY5Y cell) After performing the staining method, the ciliary morphology of the cells was confirmed through a fluorescence microscope.

As a result, as shown in FIG. 2 , it was confirmed that the number and length of primary cilia were decreased in HSD17B4 gene KO cells. In addition, as a result of confirming the amount of intracellular acetyl-CoA using the intracellular acetyl-CoA quantitative analysis kit (Sigma), it was confirmed that the amount of acetyl-CoA in KO cells was significantly reduced by about 10% or more compared to normal-expressing neuroblasts. .

<Example 3> Confirmation of ciliation and acetyl CoA levels in cells derived from a patient (PBD) having a HDD17B4 mutant gene

Human-derived fibroblasts (fibroblast, Control, GM00969, Coriell) with normal expression of HSD17B4 gene and fibroblast cell line (GM13263, Patient) cells expressing HSD17B4 mutant gene (422del2) were prepared, and then immunized in the same manner as in the previous experiment. Fluorescence staining and intracellular acetyl-CoA quantitative analysis were performed to confirm cell ciliation and intracellular acetyl-CoA levels, respectively.

As a result, as shown in FIG. 3 , it was confirmed that primary ciliary ciliated cells and lengths of cilia were significantly decreased in the patient-derived fibroblast cell line (GM13263) in which the function of HSD17B4 was suppressed compared to that of the GM00969 normal cell line, and intracellular acetyl- It was confirmed that CoA was also significantly reduced.

<Example 4> Confirmation of cilia length change by acetyl-CoA in HSD17B4 knockout cells

The previously prepared SH-SY5Y/Cas9 cell line (WT) and HSD17B4 knockout SH-SY5Y/Cas9 HSD17B4 KO (KO) cell line were treated with acetyl-CoA (100 μM, Sigma) and octanoyl-CoA (100 μM, Sigma) for 24 hours. After that, the cells were washed with normal culture medium, and immunohistochemistry and intracellular acetyl CoA quantitative analysis were performed.

As a result, as shown in FIG. 4 , the number of ciliated cells with primary cilia, which were reduced by HSD17B4 knockout, and the length of primary cilia were restored to more than normal levels by acetyl-CoA treatment, acetyl-CoA and octanoyl-CoA It was confirmed that the amount of acetyl-CoA, which was reduced by HSD17B4 knockout, was restored by the treatment.

<Example 5> Confirmation of ciliary abnormality recovery effect by acetyl-CoA in PBD patient-derived fibroblasts

In the same manner as in the previous experiment, normal human-derived fibroblast cell line (Cont) and HSD17B4 gene mutation patient (PBD)-derived fibroblast cell line (patient) were treated with 100 μM of acetyl-CoA and octanoyl-CoA for 24 hours. Changes in ciliation and the amount of acetyl-CoA were confirmed.

As a result, as shown in FIG. 5 , the level of ciliated cells in PBD patient-derived fibroblasts increased to more than a normal level, and the amount of acetyl-CoA, which was decreased in PBD patient-derived cells by treatment with acetyl-CoA and octanoyl-CoA, was restored again. was able to confirm that

<Example 6> Confirmation of recovery of intracellular cilia-associated signaling by acetyl-CoA in HSD17B4 knockout cells

After treatment with acetyl-CoA (100 μM, 24 h) in the previously prepared SH-SY5Y/Cas9 cell line (WT) and HSD17B4 KO SH-SY5Y cell line, the target of TGF-β signaling in the intracellular signaling system associated with primary ciliary function The effect on phosphorylation of phosphorylation of SMAD 2/3 and the phosphorylation of AKT protein involved in the survival response to extracellular signals was confirmed by performing Western blotting.

As a result, as shown in FIG. 6, phosphorylation of AKT and SMAD2/3 proteins was greatly reduced in the KO cell line, but it was confirmed that phosphorylation of AKT and SMAD2/3 was normal in the acetyl-CoA-treated cell group.

From the above results, it was confirmed that acetyl-CoA treatment restored the function of primary cilia even in the HSD17B4 knockout cell line.

<Example 7> Confirmation of neuronal apoptosis inhibitory effect of acetyl-CoA and octanoyl-CoA

In order to induce a Parkinson's disease cell line model, the SH-SY5Y cell line was co-treated with the mitochondrial toxin MPP ⁺ (2.5 mM) and acetyl-CoA (100 μM) or octanoyl-CoA (100 μM) for 24 hours. After each 24 hours, cells were collected and Western blotting was performed using an antibody (cleaved caspase-3) that recognizes caspase-3 activity, an apoptosis marker.

As a result, as shown in FIG. 7 , it was confirmed that the caspase-3 activity increased in the human neuroblastoma cells induced in the Parkinson's disease model by MPP + treatment was effectively reduced by acetyl-CoA and octanoyl-CoA treatment.

From the above results, it was confirmed that acetyl-CoA and octanoyl-CoA can effectively inhibit apoptosis of neurons by MPP ⁺ treatment.

<Example 8> Confirmation of the effect of acetyl-CoA on neuronal cell death according to inhibition of primary cilia activity

Human neuroblastoma cells (SH-SY5Y cells) were transfected with siRNA for IFT88, a major regulator involved in primary cilia formation, to suppress IFT88 expression, and then MPP ⁺ acetyl-CoA and octane Oil-CoA was treated simultaneously with each. After 24 hours of treatment, cells were collected and CCK-8 assay was performed to confirm cell viability.

As a result, as shown in FIG. 8 , it was confirmed that the cell activity decreased by the MPP ⁺ treatment was further reduced in the siIFT88 group compared to the control Sc group, whereas the cell activity decreased by the treatment with acetyl-CoA and octanoyl-CoA was restored.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA), and acetyl-CoA precursor containing at least one of the group consisting of an active ingredient for preventing or treating a ciliary disease pharmaceutical composition. The pharmaceutical composition for preventing or treating ciliary disease according to claim 1, wherein the acetyl-CoA precursor is selected from the group consisting of acetate, pyruvate and citrate. The method according to claim 1, wherein the ciliary abnormal disease is peroxisome biogenesis disorders caused by primary ciliary abnormalities, neurodegenerative diseases, cancer diseases, and obesity, characterized in that selected from the group consisting of preventing or A therapeutic pharmaceutical composition. The method according to claim 3, wherein the ciliary disease is polycystic kidney disease (PKD), infertility, developmental disorder, microcephaly, facial dysmorphism, polydactyly ), visceral inversus, cognitive defect, loss of hearing and vision, retinopathy, hypotension, fibrosis A pharmaceutical composition for preventing or treating ciliary disease, characterized in that it is selected from the group consisting of. The method according to claim 3, wherein the peroxisome biogenesis disorders are Zellweger syndrome, rhizomelic chondrodysplasia punctata, β-oxidation enzyme defects, Refsum's disease (Infantile Refsum Disease) and neonatal adrenoleukodystrophy (neonatal adrenoleukodystrophy, NALD), characterized in that selected from the group consisting of ciliary abnormal disease prevention or treatment pharmaceutical composition. The method according to claim 3, wherein the neurodegenerative disease is Alzheimer's disease (Alzheimer disease), dementia (Dementia), Parkinson disease (Parkinson disease), Huntington disease (Huntington disease), amyotrophic lateral sclerosis (ALS) and memory loss from the group consisting of A pharmaceutical composition for preventing or treating ciliary disease, characterized in that it is selected. The method according to claim 1, wherein the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and the acetyl-CoA precursor increases the length of the primary cilia and the number of ciliary cells. A therapeutic pharmaceutical composition. The method according to claim 1, wherein the acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursors increase the phosphorylation of AKT and SMAD2/3 in HSD17B4 expression-suppressing cells to prevent ciliary-associated signaling A pharmaceutical composition for preventing or treating ciliary disease, characterized in that it restores it. Acetyl-CoA (Acetyl-CoA), octanoyl-CoA (Octanoyl-CoA) and acetyl-CoA precursor health food for preventing or improving ciliary disease containing at least one active ingredient as an active ingredient. treating a candidate substance in a cell line in which expression of a β-oxidation activity-regulating gene is suppressed;
identifying one or more levels of increase in the group consisting of the length of primary cilia and the number of ciliated cells in the cell line treated with the candidate substance; and
A method of screening for a therapeutic agent for ciliary abnormalities comprising comparing the increase in the length of the primary cilia and the number of ciliated cells with a normal control group. The method according to claim 10, wherein the cell line in which the expression of the β-oxidation activity regulatory gene is suppressed has reduced primary cilia length and number of ciliated cells. The method according to claim 10, wherein the β-oxidation activity regulatory gene is selected from the group consisting of HSD17B4, ABCD1, ACOX1, CACT and TYSND1.

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