KR102107061B1 - Composition for inhibiting progerin-lamin A binding comprising WRN recombinant proteins - Google Patents

Abstract

The present invention relates to a composition for inhibiting progerin-lamin A binding including WRN recombinant protein as an active ingredient. The WRN recombinant protein is shown to have an excellent effect of inhibiting progerin expression and progerin-lamin A binding in the cells derived from patients suffering from Hutchinson Gilford progeria syndrome (HGPS) or Werner syndrome. Thus, the composition can be used effectively for treating aging diseases caused by progerin-lamin A binding. In addition, the WRN recombinant protein is shown to enhance production of collagen of skin cells. Thus, the composition can be provided as a cosmetic composition for preventing or alleviating wrinkles.

Description

Composition for inhibiting progerin-lamin A binding comprising WRN recombinant proteins}

The present invention relates to a composition for inhibiting progerin-lamin A binding containing a WRN recombinant protein as an active ingredient and uses thereof.

As the lifespan of humans increases, interest in the progress of aging has been actively raised, but there are still many undiscovered areas, and recent studies have been conducted on genetic or molecular aging mechanisms mainly for human premature ejaculation. have.

Premature ejaculation or Hutchinson Gilford progria syndrome (HGPS) is a fatal and rare genetic disorder in which premature aging occurs in young children. In children with premature ejaculation, it appears normal in early infancy, but about 9-24 months When this happens, it begins to show a serious growth delay, and eventually shows a short stature and weight loss. It has a characteristic face shape, systemic atherosclerosis, cardiovascular disease, stroke, hip dislocation, etc., fat layer under the skin is lost, nail defects, joint hardness, and skeleton damage. Patients with premature digestion usually die from 8-21 years of age due to heart disease and have an average life expectancy of 13 years.

HGPS is a very rare autosomal dominant genetic disease caused by the silent mutation of G608G of Lamin A (LMN A). This mutation creates a new cleavage donor site and produces a selective cleavage site product, Progerin (Prg), which is deleted of 50 amino acids of the C-terminal domain of Lamin A.

The expression of progerin causes morphological changes such as nuclear membrane irregularity or a decrease in nuclear-cytoplasmic lamin A. When inhibition of progerin expression leads to a decrease in nuclear transformation, it has been identified as a major factor in HGPS.

Accordingly, there is a method of relieving progenin by inhibiting farnesylation of progenin as a treatment for premature ejaculation, or by removing panesylated lamin A using autophage, but in both cases side effects There are no reports on the underlying treatment of premature ejaculation.

Republic of Korea Patent Publication No. 10-2012-0106286 (released on September 26, 2018)

The present invention provides a composition containing WRN recombinant protein as an active ingredient to inhibit the binding of progerin and lamin A, and the composition is a pharmaceutical composition for preventing or treating aging-related diseases such as premature ejaculation and a cosmetic composition for improving wrinkles Want to provide.

The present invention provides a composition for inhibiting progerin-lamin A binding containing a WRN recombinant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating aging-related diseases containing a WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving wrinkles containing WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

WRN recombinant protein according to the present invention confirmed that it shows excellent progerin expression in cells derived from patients with Hutchinson Gilford progria syndrome (HGPS) or Warner syndrome, and inhibits the binding of progerin and lamin A Accordingly, it can be effectively used in the treatment of aging diseases caused by the combination of progerin and lamin A, and as the WRN recombinant protein is found to increase collagen production in skin cells, it can be provided as a cosmetic composition for preventing or improving wrinkles.

1 is a result of confirming the expression of a similar gene between HGPS and WS, and FIG. 1A shows Human Gene 1.0 ST arrays (for confirming gene expression of aging and young normal fibroblasts and progeroid syndrome cells (HGPS and WS)) Affymetrix) microarray analysis results, FIG. 1B confirmed that a significant portion of the gene showing an expression difference in which the expression of genes is increased or decreased by at least 2 times or more compared to normal human fibroblasts overlaps between HGPS and WS. Results, FIG. 1C is a result of confirming that the gene for cell cycle regulation and chromosome separation is included in the overlapping gene, and FIG. 1D is RT- for confirming the decrease of Rad51, CENP1 and cell cycle related genes in HGPS and WS cells. PCR (left panel) and WB analysis (right panel) results, FIG. 1E shows 285 commonly regulated in progesteroid cells and aged fibroblasts. As a result of confirming the former (center gene set), FIG. 1F shows that the down-regulated gene in the central gene set is associated with cell adhesion, while the up-regulated central gene set includes cell cycle, chromosome separation, DNA replication and DNA repair. As a result of confirming that related genes are included, gene clustering is a gene interaction mapping tool www. cbioportal. org.
2 is a result of confirming that the human-specific overlap region is important for aging, FIG. 2A does not require the ^progestoid phenotype of mouse WRN, Lmna ^{+ / G609G} and Lmna ^{+ / G609G} ; Wrn ^-/- as well as the results of confirming that there is no weight difference between wrn ^{+ / +} and wrn ^-/- mice, and FIG. 2B shows the effect of progerin expression on lifespan, Lmna + / G609G and Lmna + / G609G ; Wrn ^{- / -} as well as wrn ^{+ / +} and wrn ^{- / -} it is the result confirming that the lifespan difference does not appear between the mice. Figure 2C is a result of performing amino acid sequence analysis between human and mouse WRN, confirming that 28 amino acids are repeated in human WRN, and Figure 2D is a recombinant single (R1-GST) peptide using a protein delivery system and As a result of transfection of the repeating peptide (R2-GST) into WS cells, respectively, the result of confirming a decrease in p16 / INK4A expression in WS fibroblasts to which the repeating sequence of hWRN (R2) was delivered, and FIG. 2E is through R2-GST transport. It is a result of confirming that H3K9me3 expression is induced in WS cells.
3 is a result of confirming a sequence that is specifically repeated in human WRN compared to a mouse, and FIG. 3A is a detailed amino acid search result, confirming that 28 amino acids (black underlined portions) are repeated in a human sequence (red underlined portions). As a result, FIG. 3B is a cDNA analysis result confirming that the coding cDNA sequence is also copied from the repeated amino acid sequence.
Figure 4 is a result confirming the effect of improving the aging characteristics of the Warner Syndrome cells of the recombinant protein, Figure 4A is a result of inducing expression of H3K9me3 in HGPS cells transfected with human WRN (hWRN), 48 as a vector to the cells After time transfection and fixation, the result was stained with H3K9me3 antibody, and FIG. 4B shows the effect of human-specific overlapping regions on progerin-WRN binding, bead-bound GST WRN-R1 (non-repeated peptide) And WRN-R2 (duplicated peptide) was incubated with GFP-LmnA or progerin-transfected 293 cell lysate, and after pull-down analysis by centrifugation, the bound GFP protein was analyzed by Western blot, resulting in human-specific overlapping regions. As a result of confirming that progerin-WRN binding has an important effect, FIG. 4C shows that WRN-R2 blocks progerin interaction with lamin A, and bead-bound GST-progerin As a result of incubation with 293 cell lysate transfected with GFP-ramin A in the presence or absence of WRN-R1 or WRN-R2, as a result, in the experimental group to which recombinant WRN-R2 was added, lamin A-progerin As a result of confirming that the interaction is clearly reduced, FIG. 4D shows that the recombinant WRN-R2 peptide induces H3K9me3 and shows an effect of improving nuclear transformation, using a protein transporter, WRN-R1 and WRN-R2 peptide was transfected into HGPS cells for 24 hours, and WRN-R2 was confirmed to increase H3K9me3 expression and improve nuclear shape in cells carried, and FIG. 4E shows progerin in the cells of Warner syndrome (WS) patients. As a result of confirming the induction of H3K9me3 expression by removal, when progerin is knocked down using siRNA in WS cells, as a result of confirming that H3K9me3 is induced and the nuclear size is reduced, si-control (nontarget sequence) or si-ph This is the result of 48 hours transfection of logerin and staining with H3K9me3 antibody and DAPI.
5 is a result confirming that progerin is a causative factor of Warner's syndrome (WS), and FIG. 5A is a result of showing that H3K9me3 expression was induced in HGPS cells transfected with human WRN (hWRN), and vectored cells. After transfection and fixation for 48 hours, the result was stained with H3K9me3 antibody, and FIG. 5B is a result of confirming that WRN is strongly bound to progerin than lamin A, and FIG. 5C is a strong interaction between human WRN and progerin that does not appear in mouse WRN. As a result of confirming the action, FIG. 5D shows that WRN-R1 and R2 peptides are transfected into HGPS cells for 24 hours using a protein carrier, and recombinant WRN-R2 peptides induce H3K9me3 and nuclear forms in cells carrying WRN-R2. 5E is a result of staining WS cells transfected with si-control or si-progerin for 48 hours with H3K9me3 antibody and DAPI (nucleus), showing Warner's disease. In the group of patients cells was verified by H3K9me3 derived according to the procedure gerin removed, pro gerin knocked down using siRNA is the result confirming the induction and reduction in nuclear size WS cells of H3K9me3.
FIG. 6A is a result of confirming that collagen 1A expression is improved in HaCaT cells treated with a progerin inhibitor, and is analyzed by Western blot after incubating HaCaT cells and a progerin inhibitor for 24 hours, and FIG. 6B is a normal fibroblast. (GM 00038, 9-year-old female, AG13299, 46-year-old male) and progerin inhibitor were cultured for 24 hours and Western blot was performed to confirm the increased collagen 1A expression.

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

The inventors of the present invention predict that the expression of progestin increases after aging in the cells of patients with Warner syndrome, known as early aging syndrome, and are expected to be similar to the representative aging disease Hutchinson-Gilford Progeria syndrome (HGPS) As a result of comparing the genes of HGPS and Warner's syndrome (WS), it was confirmed that the genes involved in both aging diseases are very similar, and the protein having a repetitive amino acid sequence in human Warner's syndrome cells inhibits the binding of progerin and lamin A By confirming the effect of improving the progestin-related aging syndrome, the present invention was completed.

The present invention can provide a composition for inhibiting progerin-lamin A binding containing a WRN recombinant protein composed of an amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

The WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 may be a human-derived WRN amino acid sequence, and more preferably, a human-derived WRN amino acid sequence may be repeated twice.

The present invention can provide a pharmaceutical composition for preventing or treating aging-related diseases containing a WRN recombinant protein composed of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

The WRN recombinant protein may be to inhibit the binding between progerin and lamin A.

The aging-related disease may be premature ejaculation, and the premature ejaculation may be selected from the group consisting of Werner syndrome and Hutchinson Gilford progria syndrome.

In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. Can be used.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, commonly used in the manufacture of a pharmaceutical composition, It may further include at least one additive selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, 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 dosage forms for oral administration include tablets, pills, powders, granules and capsules. Agents and the like, and such solid preparations may be prepared by mixing at least one excipient in the composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

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

The preferred dosage of the compound represented by Formula 1 may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention is not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, and more specifically 0.1 to 100 mg / kg. The administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

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

In addition, the present invention can provide a cosmetic composition for preventing or improving wrinkles containing the WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.

The WRN recombinant protein may enhance collagen production in keratinocytes and fibroblasts.

The cosmetic composition may include a conventional adjuvant such as a stabilizer, a solubilizing agent, vitamins, pigments and flavors, and a carrier in addition to WRN recombinant protein as an active ingredient.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, oils, powder foundations, emulsion foundations, It may be formulated as a wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of a sun cream, soft lotion, converging lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, pack, spray or powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide may be used as a carrier component. .

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of a spray, additionally chlorofluorohydrocarbon, propane / butane Or propellants such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Fatty acid esters of butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or trakant, and the like.

Hereinafter, examples will be described in detail to help understanding of the present invention. 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 fully describe the present invention to those skilled in the art.

< Experimental Example  1> Cell culture and reagent

HGPS (Hutchinson Gilford progeria syndrome) patients (AG03198, 10-year-old female; AG03199; 10-year-old female), WS (werner syndrome) patients (AG06300, 37-year-old male; AG03141, 30-year- Human fibroblasts of old female; AG00780, 60-year-old male) and control (GM 00038, 9-year-old female) were obtained from Coriell Cell Repositories (Camden, New Jersey, USA), 15% FBS, It was maintained in EMEM medium containing 2 mM glutamine or HEMEM containing 26 mM HEPES without antibiotics.

The HEK293 cell line was obtained from ATCC and maintained at 37 ° C. using DMEM liquid medium containing 10% FBS and 1% antibiotic.

< Experimental Example  2> Antibodies and reagents

GFP (1: 1000; sc-9996; Santa Cruz Biotechnology); GST (1: 5000; sc-138; Santa Cruz Biotechnology); Actin (1: 10000; sc-47778; Santa Cruz Biotechnology); Lamin A / C (1: 10000; sc-376248; Santa Cruz Biotechnology); Progerin (1: 300; sc-81611; Santa Cruz Biotechnology); Progerin (1: 300; ab66587; Abcam); Antibodies such as H3K9me3 (1; 2000; Ab8898; Abcam) and p16-INK4A (1: 500; 10883-1-AP; Proteintech) were used in the experiment.

< Experimental Example  3> WRN  Recombinant protein

For the production of recombinant proteins, 100 AA was cloned from the upstream of the termination codon through PCR to produce recombinant lamin A C-terminal region (Lamin AC) and progerin C-terminal region (Progerin C), and WRN-R1 region ( hWRN 424-450) and WRN-R2 regions (hWRN 424-476) were produced in a similar manner.

After loading GSH-agarose to each fragment, it was washed extensively and eluted using a buffer containing 20 mM reduced glutathione.

The eluted fragment was further purified using anion exchange chromatography (HitrapQ) to obtain the following WRN-R1 and WRN-R2 amino acid sequences.

WRN-R1: HLSPNDNENDTSYVIESDEDELEMEMLK (SEQ ID NO: 1)

WRN-R2: HLSPNDNENDTSYVIESDEDELEMEMLK HLSPNDNENDTSYVIESDEDELEMEMLK (SEQ ID NO: 2)

< Experimental Example  4> Western Blot  analysis

Whole cell lysates were prepared using RIPA.

15 μg of cell extract was separated on SDS-PAGE and transferred onto PVDF membrane.

The membrane was incubated with the primary antibody for 1 hour to 4 ° C. overnight, and then reacted for 1 hour at room temperature using the secondary antibody.

Peroxidase activity was confirmed by chemiluminescence according to the manufacturer's instructions using the ECL kit (Intron, Seoul, Korea).

< Experimental Example  5> Protein-protein interaction analysis

For protein-protein interaction analysis, glutathione S-transferase (GST) immune pull-down analysis was performed.

For interaction detection, GST-based fused lamin AC-terminal region, progerin-C-terminal region, WRN-R1 region or WRN-R2 region and GFP-tagged progerin (GFP-Progerin) and lamin A (GFP- HEK293 cell lysates transfected with Lamin A) were incubated at room temperature for 30 minutes.

Then, it was washed once with PBS, and the precipitated material was collected and separated on SDS-PAGE, followed by western blot with anti-GFP and GST.

In order to confirm the competitive response of WRN-R1 and R2 to progerin and lamin A binding, GFP-ramine A was used for GST-progerin bound beads in the presence or absence of WRN-R1 or R2 recombinant protein. Incubated with overexpressed 293 lysate.

< Experimental Example  6> Immunofluorescence  Staining and aging specific oxidation β-galactosidase active staining

Cells were inoculated onto the cover glass, the appropriate vector was transfected, and fixed at 4 ° C for 1 hour using 100% methanol or 1% paraformaldehyde (PFA), and then the cells were blocked for 1 hour with blocking buffer (PBS + anti- human-Ab; 1: 400).

After washing twice with PBS, anti-lamin A / C, progerin or H3K9Me3 was diluted 1: 200 in blocking buffer overnight to incubate with the cells and subsequently anti-goat Ab-FITC or anti-rabbit Ab- Incubated with blocking buffer (rhodamin) containing rhodamin (1: 500) for 7 hours, and nuclei were stained with DAPI.

Thereafter, the immunofluorescence signal was detected using a fluorescence microscope (Zeiss and Logos).

To stain the aging specific oxidation-β-galactosidase activity, cells were washed with PBS (pH 7.2) and fixed with PBS containing 0.5% glutaraldehyde. Thereafter, the cells were washed with PBS and stained with X-gal solution (9860; Cell Signaling Technology) at 37 ° C overnight.

< Experimental Example  7> RNA isolation and RT- PCR

To perform RT-PCR, whole cell RNA was extracted using an RNA extraction kit (Qiagen). After confirming the RNA concentration, 1 μg of total RNA was reverse transcribed into cDNA using MMLV RT (Invitrogen) and random hexamers.

RT-PCR was performed using the following specific primers: Rad51, 5'-TCTTTGGTTTTGGAGGAGGGG-3 'and 5'-GTAGGTTTGGCACAAGACTCC-3'; DCR2, 5'-ACTGGGAAAACCCCAGCAGCG-3 'and 5'-TTCCAGCAGACGCTGTGGCTC-3'; GAPDH 5'-ATCTTCCAGGAGCGAGATCCC-3 'and 5'-AGTGAGCTTCCCGTTCAGCTC-3'.

< Experimental Example  8> Plasmid transfection and protein delivery

GFP-progerin and GFP-fusion lamin A expression vectors were provided by T. Misteli (National Cancer Institute [NCI], Bethesda, Maryland, USA), Myc-human WRN vector and Myc-mouse WRN vector were purchased from Addgene. . Transfection was performed according to the manufacturer's instructions using jetPEI (Polyplus Transfection) and PULSin (Polyplus Transfection, New York, USA).

To deliver WRN-R1 and R2 proteins into HGPS cells, PULSin (Polyplus Transfection, New York, USA) was used according to the manufacturer's instructions.

After diluting the recombinant protein (2 μg) with 200 μl of 20 mM Hepes, 8 μl of PULSin reagent was added and the mixture was incubated for 15 minutes at room temperature.

After the incubation, the mixture was added to the cells, and after 3 hours, the serum-free medium was replaced with a medium containing 10% fetal bovine serum and incubated for 4 hours.

Thereafter, the medium containing mixture was removed from the wells and fresh medium containing serum was filled.

< Experimental Example  9> Cell count

To count cells with malformed nuclei, the obtained immunofluorescence image was used. Nuclear membranes that appeared to be malformed dependent on Lamin A staining in randomly selected zones were counted and the counted cells expressed as percentages.

In addition, DAPI stained cells were counted in an immunofluorescence image to confirm cell proliferation.

< Experimental Example  10> Micro Array Analysis

500 ng of total RNA was extracted using RNAeasy kit (Qiagen).

RNA labeling, Human Gene 1.0 ST arrays (Affymetrix) hybridization and data analysis were performed on DNA Link (Seoul, Korea (rep. Of)).

Genes showing at least 2 fold differences in either cell line were selected for further analysis.

< Experimental Example  11> Statistical analysis and protein network analysis

To confirm the statistical significance of the observed differences, a student's t-test was conducted. For protein network analysis, 100 genes were collected from the HARG / JR set, and a network program in cBioportal package (http://www.cbioportal.org) was performed.

< Experimental Example  12> Chemical pharmacokinetic analysis

For pharmacokinetic (PK) analysis, 10% DMSO, 5% Tween 90 and 95% physiological saline solution in which JH4 was dissolved at 5 mg / kg was injected intraperitoneally, and 10% NMP and 90% PEG400 solution in which JH4 was dissolved at 10 mg / kg was added. It was administered orally.

The blood concentration of JH4 was analyzed by LC-MS / MS every predetermined time, and other compounds were also PK analyzed in the same process.

In vitro ADME studies (plasma protein binding, CYP inhibition, microsomal stability, plasma stability and hERG inhibition) were performed in a new drug development center by standard methods.

< Example  1> human origin WRN  Of recombinant protein Progestin - Lamin  A Confirmation of binding inhibitory effect

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disease known as premature ejaculation, and it is known that lamin A mutated by abnormal splicing causes progerin, a previous study. The report confirmed that the nuclear abnormality was due to a very strong bond between lamin A and progestin.

In addition, Warner syndrome (Werner syndrome) is known as an early aging syndrome, an aging disease that has not been studied for treatment, and since progesterine expression increases after aging, it is expected to be similar to HGPS. Microarray analysis was performed to compare HGPS and Warner syndrome (WS).

As a result, as shown in Figure 1A, it can be confirmed that Warner syndrome is very similar to the gene expression profile of HGPS cells.

Referring to FIG. 1B, more than 60% of the WS-specific gene (881 gene) overlaps with the HGPS-specific gene, and the genes are induced into HGPS with genes involved in cell cycle, DNA replication, and chromosome separation as shown in FIG. 1C. It has also been reported to be associated with aging.

In addition, referring to Table 1 and Table 2, it was confirmed that HGPS exclusive genes are related to cell adhesion or ECM, and they are not directly related to aging, but WS exclusive genes are related to cell cycle and somatic cell division. Western blot analysis (WB) and RT-PCR analysis were performed to confirm the microarray results as shown in FIG. 1D.

From the above results, 285 genetic modifications were confirmed in aged fibroblasts among 881 genes, which are common genes between HGPS and WS, as shown in FIG. 1E. In addition, it was confirmed that the majority of the aging-related central gene set overlaps with the common aging gene of HGPS as shown in FIG. 1F, and the genes generally down-regulated in HGPS, WS, and aging fibroblasts are cell cycle, DNA replication, and chromosomal division. It was confirmed to be related to.

On the other hand, the up-regulated gene set is involved in cell adhesion, and these characteristics are very valid because aging induces cell spreading and adhesion and cell cycle arrest, thus confirming the effect of the Warner syndrome WRN gene in the HGPS mouse model.

According to previous reports, the weight and lifespan of WRN knocked-out mice did not show a distinct difference from wild-type mice (Lombard, DB et al., Mol . Cell. Biol . 20, 3286-3291, 2000; Aumailley L. et al ., PLoS One 10 , e0140292, 2015), between mWRN ^-/- as shown in FIG. Differences in weight and lifespan of the Lamn ^{wt / G609G} and Lamn ^{wt / G609G} mice were not confirmed.

However, as a result of comparing the amino acid sequence of mouse and human WRN, a sequence that is specifically repeated in human WRN (WRN-R2) as shown in FIGS. 2C and 3 was confirmed, and in fact, overlap of hWRN was generated by repeated cDNA.

In order to confirm the importance of the repeat sequence in WS, a WRN-R1 consisting of 28 amino acids and a WRN-R2 recombinant peptide consisting of 56 amino acids having a repeat sequence were prepared in the same manner as in Experimental Example 3, and the recombinant WRN- in WS cells. R1 or WRN-R2 was transfected.

As a result, as shown in FIG. 2D, p16 / INK4A expression was decreased in WS cells transfected with WRN-R2, and as WRN-R2 was delivered to WS cells, it was confirmed that H3K9me3 was induced as shown in FIG. 2E. .

In addition, to confirm the effect of WRN on HGPS cells, human and mouse WRN were transfected into HGPS cells, and nuclear morphology and H3K9me3 expression were confirmed.

As a result, as shown in FIGS. 4A and 5A, nucleation and relaxation of h3K9me3 were induced in hWRN.

On the other hand, as a result of confirming the interaction between progerin and WRN, the interaction between WRN and progerin was stronger than that between WRN and lamin A as shown in FIG. 5B, and hWRN and pro were not shown in mouse WRN as shown in FIG. 5C. Guerin's strong interaction was confirmed.

From these results, it was predicted that a human specific repeat region would be responsible for strong binding between progerin and WRN.

To confirm this, GST-immune sedimentation analysis was performed using recombinant WRN-R1 and WRN-R2, and as a result, WRN-R2, a double peptide, compared to WRN-R1 as shown in FIG. In fact, as shown in Figure 4C, WRN-R2 showed the effect of blocking the interaction of progerin and lamin A.

In addition, the delivery of recombinant WRN-R2 to HGPS cells normalizes nuclear morphology as shown in FIG. 4D, improves H3K9me3 expression, and shows good effects through progesterine removal in WS cells as shown in FIGS. 4E and 5E. I could confirm.

From the above results, it was confirmed that WRN-R2 is a natural progerin inhibitor (Progerinin; Progerin inhibitor).

< Example  2> Person origin WRN  Confirmation of wrinkle improvement effect of recombinant protein

Meanwhile, the effect of progerin inhibitors on human skin keratinocytes and fibroblasts was confirmed.

The progerin inhibitor was transfected into HaCaT cells and normal fibroblasts, cultured for 24 hours, and then Western blot analysis was performed.

As a result, as shown in FIGS. 6A and 6B, it was confirmed that the progerin inhibitor increases collagen and fibronectin expression in human fibroblasts and keratinocytes.

From the above results, it was confirmed that the progestin inhibitor WRN-R2 can be used not only for WS syndrome but also as a potent candidate drug for HGPS and as a composition for improving wrinkles against aging skin.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> PRG S & Tech Inc. <120> Composition for inhibiting progerin-lamin A binding comprising          WRN recombinant proteins <130> DP-2018-0242 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 28 <212> PRT <213> WRN-R1 <400> 1 His Leu Ser Pro Asn Asp Asn Glu Asn Asp Thr Ser Tyr Val Ile Glu   1 5 10 15 Ser Asp Glu Asp Glu Leu Glu Met Glu Met Leu Lys              20 25 <210> 2 <211> 56 <212> PRT <213> WRN-R2 <400> 2 His Leu Ser Pro Asn Asp Asn Glu Asn Asp Thr Ser Tyr Val Ile Glu   1 5 10 15 Ser Asp Glu Asp Glu Leu Glu Met Glu Met Leu Lys His Leu Ser Pro              20 25 30 Asn Asp Asn Glu Asn Asp Thr Ser Tyr Val Ile Glu Ser Asp Glu Asp          35 40 45 Glu Leu Glu Met Glu Met Leu Lys      50 55

Claims (8)

A reagent composition for inhibiting progerin-lamin A binding comprising a WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient. The method according to claim 1, WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is a reagent composition for inhibiting progerin-lamin A binding, characterized in that the human-derived WRN amino acid sequence is repeated. A pharmaceutical composition for preventing or treating premature ejaculation, comprising a WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient. The method according to claim 3, wherein the WRN recombinant protein is a pharmaceutical composition for preventing or treating premature ejaculation, characterized by inhibiting the binding between progerin and lamin A. delete The method according to claim 3 or 4, wherein the premature ejaculation is Werner syndrome (Werner syndrome) and Hutchinson Gilford syndrome (Hutchinson Gilford progria syndrome) pharmaceutical composition for preventing or treating premature ejaculation, characterized in that selected from the group consisting of. A cosmetic composition for preventing or improving wrinkles containing WRN recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient. The method according to claim 7, WRN recombinant protein is a cosmetic composition for preventing or improving wrinkles, characterized in that to enhance collagen production in keratinocytes and fibroblasts.

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