KR100789848B1 - Method of inhibiting apoptosis by inhibition of FLASH activity - Google Patents

Abstract

The present invention is to suppress the activity of FLASH (FLICE-associated huge protein) to inhibit apoptosis of cells, more specifically, the inhibition of FLASH protein activity is complementary to the peptide aptamer or mRNA of FLASH Through an activity inhibitor comprising antisense nucleotides. The FLASH protein aptamer of the present invention inhibits TNF-α-derived apoptosis, and FLASH antisense nucleotides effectively inhibit TNF-α-derived and Fas-derived apoptosis to treat stroke or neurological diseases in which apoptosis occurs excessively. It can be useful for development.

Peptide Aptamers, Antisense Nucleotides, FLASH, Apoptosis

Description

Method of inhibiting apoptosis by inhibition of FLASH activity

1 is a conceptual diagram of an apoptosis process involving FLASH.

Figure 2 shows the gene of FALSH.

FIG. 3 shows the binding of selected peptide # 1 to FALSH-DRD:

A: measuring binding force with peptide # 1;

    Concentrations of peptide # 1 from the top were 1,000 μM, 500 μM, 250 μM, 100 μM, 50 μM, respectively;

B: determination of this binding force with the control peptide; And

    The concentrations of the control peptide from the top were 1,000 μM and 500 μM, respectively.

Figure 4 shows the effect of peptide # 1 on the binding force between Caspase-8 and FLASH-DRD.

5 shows a process for preparing a vector expressing peptide # 1:

A: primer used for PCR; And

B: Ligation process.

FIG. 6 shows the effect of peptide # 1 aptamer on apoptosis:

A: pEGFP-C3 vector transformed Hela cell line, 0 h;

B: pEGFP-SE # 1 vector transformed Hela cell line. 0 hours;

C: pEGFP-C3 vector transformed Hela cell line, 24 h;

D: pEGFP-SE # 1 vector transformed Hela cell line, 24 h;

    Left: GFP measurement;

    Right: Hoechst staining;

E:% of cells apoptotic upon pEGFP-C3 or pEGFP-SE # 1 vector transformation; And

F: Western blot results of the expression level in the cell line during pEGFP-C3 or pEGFP-SE # 1 vector hemostasis.

FIG. 7 shows FACS irradiation of Hela cell lines transformed with pEGFP-C3 or pEGFP-SE # 1 vectors. FIG.

8 shows the effect of peptide # 1 aptamer on caspase:

A: Western blot of the activity of caspase-8 and caspase-3 when transforming pEGFP-C3 or pEGFP-SE # 1 vector,

B: Immunocytochemistry, which investigated the activated caspase-8 when transforming the pEGFP-C3 or pEGFP-SE # 1 vector,

C: Investigation of the degree of apoptosis of Hela cell line transformed with pEGFP-C3, pEGFP-SE # 1, pEGFP- # 1 Reverse vector under TNF-α treatment, and

 D: Investigation of the degree of apoptosis of Hela cell line transformed with pEGFP-C3, pEGFP-SE # 1, pEGFP- # 1 Reverse vector under etoposide treatment.

9 shows the degree of apoptosis of peptide aptamer variants:

       Mock: negative control;

       SE # 1: peptide aptamer # 1;

       18: peptide aptamer variant of SEQ ID NO: 18;

       19: peptide aptamer variant of SEQ ID NO: 19;

       20: peptide aptamer variant of SEQ ID NO: 20; And

       21: Peptide aptamer variant of SEQ ID NO: 21.

FIG. 10 shows the effect of antisense nucleotides of FLASH on apoptosis:

A: TNF-α / CHX treatment;

B: FAS ligand treatment;

C: A23187 treatment;

D: Staurosporin treatment;

E: Etoposide treatment;

F: RT-PCR;

    AS-1: antisense nucleotide; And

    AS-2: control antisense nucleotide.

The present invention relates to a method for inhibiting apoptosis of cells by inhibiting the activity of FLASH.

Multicellular individuals maintain their cell numbers by controlling cell proliferation as well as cell death. Apoptosis is caused by apoptosis, a cell suicide, which not only plays an important role in maintaining homeostasis, but also greatly affects the development and differentiation of various tissues. In addition, apoptosis is a self-defense by removing cells or cells infected with viruses that can be a threat to the individual.

Apoptosis has a morphological characteristic that distinguishes it from cell necrosis called necrosis. In necrosis, cell necrosis destroys the cell membrane, causes cell lysis and releases inflammatory factors in the cell, causing an inflammatory response. In the case of cell death due to apoptosis, the inflammatory reaction does not occur because the cells are formed by the apoptotic body and are removed by the adjacent phagocytes without the release of the intracellular material. Apoptosis occurs in a number of morphological, biochemical, and physiological features, including membrane blebbing, chromatin condensation in the nuclear membrane, formation of apoptotic bodies, Formation of nucleosomal DNA ladder, expression of phosphatidyl serine, a phospholipid of cell membrane, phagocytosis by phagocyte or macrophage (Michael, OH. Nature 407) : 770-776, 2000).

The main mechanism by which external stimulation causes apoptosis is apoptosis mediated by receptors (Nagata, S. Cell 88: 355-365, 1997). Among them, many studies have been carried out on a method for signaling cell death by Fas. Fas is a receptor that is expressed throughout the surface of various types of cells belonging to the Tumor Necrosis Factor (TNF) receptor family, and the cells expressing Fas bind to FasL (Fas ligand, CD95L), a ligand of Fas. To propagate apoptosis signals (Yonehara, S., Ishii, A., Yonehara, M., J. Exp. Med . 169: 1747-1756, 1989; Suda, T., et al ., Cell 75: 1169-1178, 1993). TNF-α is a cytokine structurally similar to FasL and performs various functions to participate in cellular defense mechanisms (Beg, A., Baltimore, D., Science 274: 782-784, 1996). TNF-α transmits signals that induce apoptosis by the receptor TNF-R and cells that survive cells through NF-κB, the opposite action. This balance control is thought to play an important role in maintaining the appropriate number of cells. First, the signaling system that causes apoptosis by TNF-α is as follows (Hsu, H., Shu, H., Pan, MG., Cell 81: 495-504, 1996; Shu, H. , Takeuchi, M., Goeddel, DV., PNAS 93: 13973-13978, 1996; Hw, W., Johnson, H., J. Biol . Chem . 275: 10838-10844, 2000). As a signal for early apoptosis, when TNF-α binds to its receptor, TNF-R1, and activates it, the death domain of TNF-R1 is associated with the Fas-Associated Death Domain protein (FADD). By dragging TRADD, procaspase-8 is cleaved into caspase-8 and activated through the DED portion of FADD (Boldin, MP, et al . , J. Biol . Chem . 270: 7795-7798, 1995; Bolddin, MP., Et al . , Cell 85: 803-815, 1996). In other words, TNF-R1, FADD, and caspase-8 form DISC ( D eath I n S ignalling C omplex) to prepare for signal transmission to the next stage (Kischkel, FC., Et . al ., EMBO J. 14: 5579-5588, 1995; Muzio, M., et al ., Cell 85: 817-827, 1996; Muzio, M., et al ., J. Biol . Chem ., 273: 2926-2930, 1998). This activated caspase-8 induces apoptosis by cleaving the caspase type in the next step and activating caspase-3, the caspase that is an executive qualification (Fernande-Alnemri, T., Litwack, G., Alnemri, ES., Cancer Res . 55: 2737-2752, 1997; Villa, P., Kaufmann, SH, Earnshaw WC., Trends Biochem . Sci ., 22: 388-393, 1997; Scaffidi C., et al ., EMBO J 17: 1675-1687, 1998). In contrast to the pathway that causes such apoptosis, the cell survival pathway by TNF-α is as follows. When TNF-R is activated by TNF-α, it binds to TRAF2 and RIP, which in turn activates NIK, which in turn activates the Mitogen-Activated Protein Kinase family, IKKα and IKKβ, resulting in the degradation of IκB due to phosphorylation of IκB. By activating NF-κB and PDK1 by PI3K phosphorylating Akt, NF-κB activates several genes involved in cell survival due to activation of cells (Wang, CY., Et . al ., Science 281: 1680-1683, 1998; Liu, Z., et al ., Cell 87: 565-576, 1996; Malinin, NL., Et al ., Nature 385: 540-544, 1997).

Recently, a protein called FLASH ( FL ICE- As sociated H protein) has been found to be a new molecule that forms DISC in apoptosis mediated by Fas or TNF-α (see FIG. 1; Imai, Y. , et al ., Nature 398: 777-785, 1999; Medema, JP., Nature 398: 756-757, 1999). E1B19K of anti-apoptotic adenovirus does not bind to Fas, FADD or caspase-8, but indirectly interferes with Fas or FADD-mediated caspase-8 activation. Based on this, it is inferred that there is a new molecule for forming DISC and activating caspase-8 (Perez, D., White, E., J. Biol. Dhem. 276: 25073-25077, 1998). T-cell cDNA library was screened through a yeast two-hybrid system using DED (Death Effector Domain) of procaspase-8 to separate the protein of the new function. In addition to FADD, which is known to bind caspase-8, FLASH has a molecular weight of 219.1 kDa consisting of 1962 amino acids with homology to DED. The FLASH is coupled with caspase-8 or DED of FADD upon forming the DISC in the DRD (D eath effector domain R ecruiting D omain) to the C- terminal. The FLASH was confirmed to be a constituent molecule of DISC that binds caspase-8 and FADD. When FLASH is transiently overexpressed, FLASH activates caspase-8 and affects apoptosis. Self-association and binding to E1B19K from the anti-apoptotic adenovirus. It has also been reported that FLASH is involved in cell survival and activates NF-κB through TRAF2 (Choi YH., Et . al ., J. Biol . Chem . 276: 25073-25077, 2001).

Accordingly, the present inventors completed the present invention by making peptide aptamers of FLASH or antisense nucleotides of FLASH to inhibit apoptosis by inhibiting FLASH activity in animal cell lines.

It is an object of the present invention to provide a method of inhibiting the activity of FLASH to inhibit apoptosis in cells.

In order to achieve the above object, the present invention provides a method for inhibiting the activity of FLASH (FLICE-associated huge protein) to suppress apoptosis (apoptosis).

The present invention also provides an apoptosis inhibitor comprising an active inhibitor of FLASH as an active ingredient.

In addition, it provides a therapeutic agent for diseases comprising the FLASH activity inhibitor as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for inhibiting apoptosis by inhibiting the activity of FLASH.

The present inventors fabricated a target molecule fused with glutathione-S-transferase (GST), named GST-FLASH-DRD (Death effector domain Recruiting Domain), and bio-panning the target molecule using a phage display method. (biopanning) was performed, and the peptide sequence showing binding to GST-PLASH-DRD was selected three times (see Table 2). The most frequent peptide sequence was named peptide # 1 (SEQ ID NO: 4) and amplified using M13 phage. The selected peptide # 1 sequence was synthesized by binding biotin to the N-terminus. Surface Plasmon Resonance (SPR) was performed to confirm the degree of binding of the synthesized peptide aptamer # 1 to GST-FLASH-DRD. As a result, it was confirmed that the binding force increased depending on the concentration (see Fig. 3A). In contrast, the control peptide was found to significantly reduce the degree of binding compared to peptide # 1 (see Figure 3B). Kd value of peptide aptamer # 1 was determined to be 5.61 × 10 ⁻⁴ M. We performed an in vitro binding assay to investigate whether the selected peptide aptamer # 1 actually inhibited the binding of FLASH-DRD and DED of caspage-8. As a result, it was confirmed that the peptide aptamer # 1 binds to FLASH-DRD and interferes with the binding of FLASH-DRD and DRD of caspage-8 (see FIG. 4).

Based on the results, in order to investigate how peptide aptamer # 1 affects apoptosis in vivo, the inventors prepared an expression vector expressing peptide aptamer # 1 (see FIG. 5). A vector expressing peptide aptamer # 1 was named "pEGFP-SE # 1" and a vector in which the gene of peptide # 1 was cloned in the opposite direction was named "pEGFP- # 1 Reverse". The pEGFP-C3 (empty vector) or pEGFP-SE # 1 vector was transformed into Hela cell lines and then treated with TNF-α, which induces apoptosis, and then examined for apoptosis. As a result, it was confirmed that apoptosis was suppressed in the cell line transformed with the pEGFP-SE # 1 vector rather than the cell line transformed with the pEGFP-C3 (empty vector) (see FIGS. 6A to E). In addition, it was confirmed that pEGFP-C3 (empty vector) and pEGFP-SE # 1 vectors stably expressed in animal cell lines (see FIG. 6F). The present inventors transformed the vector into the cell line under the same conditions, and then examined DNA damage by FACS (Fluoresence Activated Cell Sorter) to confirm apoptosis. As a result, it was confirmed that apoptosis was suppressed from 37.43% to 27.74% in the cell line expressing the pEGFP-SE # 1 vector in GFP ⁺ cells (see FIG. 7). Peptide aptamer is expressed only in GFP ⁺ cells because a peptide aptamer tagged with GFP was prepared by inserting a sequence capable of expressing the peptide aptamer of the present invention into the pEGFP vector. Since GFP ^- cells are cells that are not transformed and no peptide aptamers are expressed, GFP ⁺ cells are cells that are transformed to express peptide aptamers tagged with GFP.

Subsequently, the present inventors investigated the effect of peptide aptamer # 1 on caspase-8, which forms the Death Inducing Signaling Complex (DISC) and the caspase-3 activated by DISC. As a result, it was confirmed that peptide aptamer # 1 inhibited the activity of caspase-8 and the activity of caspasge-3 (see FIGS. 8A and B). In vivo, peptide aptamer # 1 inhibited the binding of FLASH-DRD to DED of caspage-8, and also inhibited the activity of caspasge-3, which is activated by DISC, to inhibit apoptosis in vivo. It can be seen.

We investigated the apoptosis by transforming pEGFP-C3 (empty vector), pEGFP-SE # 1, pEGFP- # 1 Reverse vectors into HeLa cell lines, and then treating TNF-α or etoposide to the cell lines. Etoposide is a Topoisomerase Ⅱ inhibitor that induces apoptosis like TNF-α, but induces DNA damage and induces apoptosis by releasing mitochondrial cytochrome C. As a result, apoptosis inhibition was observed in the cell line transformed with the pEGFP-SE # 1 vector in the TNF-α-treated group, whereas apoptosis inhibition was observed in each vector in the etoposide-treated group. Could not be observed. It can be seen that peptide aptamer # 1 is specifically inhibited by acting only on apoptosis induced by TNF-α.

In this regard, the present inventors prepared a variant of the peptide aptamer # 1 (SEQ ID NOs: 17 to 21), and prepared an expression vector to express the variant in a cell to investigate the effect on the apoptosis after transformation into the cell. It was. As a result, the variant of SEQ ID NO: 17 could not be expressed, and thus the degree of cell death could not be measured. It was confirmed that other variants inhibit apoptosis as peptide aptamer # 1 (see FIG. 9).

The present inventors confirmed that the apoptosis can be inhibited when FLASH activity was inhibited, and thus antisense nucleotides that inhibit the activity of FLASH were prepared to investigate the effect on apoptosis in cells. . As a result, it was confirmed that the FLASH peptide aptamer inhibited only TNF-α induced apoptosis but the FLASH antisense nucleotide of the present invention could inhibit not only TNF-α induced apoptosis but also Fas induced apoptosis (see FIG. 10). .

The method of inhibiting apoptosis may be performed using antisense nucleotides that complementarily bind to mRNA of FLASH.

Antisense  Nucleotide

Antisense nucleotides are currently recognized as therapeutic agents with promise in the treatment of many human diseases. Nucleotides, as defined in Watson-click base pairs, bind (hybridize) the complementary sequences of DNA, immature-mRNA or mature mRNA to disrupt the flow of genetic information as proteins in DNA. The nature of antisense nucleotides to make them specific for the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl . Cancer Inst . , 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel form of therapeutic agent because of recent advances in the field of nucleotide synthesis that exhibit oligonucleotide chemistry and improved cell adsorption, target binding affinity and nuclease resistance. For example, the use of antisense oligonucleotides targeted to cmyb has been used to completely remove myeloid leukemia cells from bone marrow derived from myeloid leukemia patients (Gewirtz and Calabreta, US Pat. No. 5,098,890). Antisense nucleotides are known to have in vivo human efficacy of treating cytomegalovirus retinitis infections. In the embodiment of the present invention, the FLASH antisense nucleotide described in SEQ ID NO: 22 was prepared and examined for the apoptosis effect. As a result, apoptosis was effectively suppressed. The FLASH antisense nucleotides according to the invention are provided to specifically hybridize with RNA derived from a gene encoding FLASH, and nucleotides with sufficient identity and number to be effective such specific hybridization are within the scope of the invention, preferably A nucleotide having a nucleotide sequence shown in SEQ ID NO: 22.

The present invention provides an apoptosis inhibitor comprising the FLASH activity inhibitor as an active ingredient. The apoptosis inhibitor of the present invention is an apoptosis inhibitor comprising a peptide aptamer of FLASH as an active ingredient, wherein the peptide aptamer preferably has an amino acid sequence set forth in SEQ ID NO: 1 or 17 to 21, It is characterized by suppressing TNF-α-derived apoptosis. In addition, the apoptosis inhibitor of the present invention is an apoptosis inhibitor comprising as an active ingredient an antisense nucleotide complementary to the mRNA of FLASH, the antisense nucleotide preferably has a nucleotide sequence as shown in SEQ ID NO: 22 And inhibiting TNF-α-derived or Fas-derived apoptosis.

The present invention provides a FLASH antisense nucleotide expression vector.

The FLASH antisense nucleotide included in the expression vector is preferably the nucleotide sequence set forth in SEQ ID NO: 22. The expression vector is not particularly limited, but it is preferable to use a conventional plasmid vector or a viral vector, and a viral vector such as an adenovirus, an adeno-associated virus, a retrovirus including a lenti virus, and the like may be used. have.

The present invention provides an apoptosis inhibitor comprising the antisense nucleotide expression vector of FLASH as an active ingredient. The apoptosis inhibitor is characterized by inhibiting TNF-α derived and Fas derived apoptosis in cells.

The composition of the present invention provides a disease treatment agent comprising the apoptosis inhibitor as an active ingredient.

 The disease treatment agent of the present invention is directed to a disease consisting of a group consisting of stroke, Alzheimer's disease, Parkinson's disease or Lou Gehrig's disease.

The composition of the present invention comprises 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

Nucleotides or nucleic acids used in the present invention can be prepared for the purpose of topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like. Preferably, the nucleic acid or vector is used in injectable form. Thus, in particular, the area to be treated may be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. The composition may comprise, in particular, an isotonic sterile solution or a lyophilized composition which allows for the composition of an injectable solution upon addition of sterile water or suitable physiological saline solution. Direct injection of nucleic acid into the patient's disease site is advantageous because it allows the treatment efficiency to focus on the infected tissue. The dosage of nucleic acid used can be adjusted by various parameters, in particular by gene, vector, mode of administration used, disease in question or alternatively desired duration of treatment. In addition, the range varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the patient. The daily dosage is about 0.01 to 12.5 mg / kg, preferably 1.25 to 2.5 mg / kg, preferably administered once to several times a day.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 FLASH ( FLICE - associated Huge protein ) -DRD ( Death effector Bio Penning (biopanning) for the domain Recruiting Domain)

< Example  1-1> FLASH - DRD  Produce

We used GST-FLASH-DRD as the target molecule for phage display. FLASH-DRD used in the present invention is fused with GST, DRD has a molecular weight of about 30 kDa (Fig. 2). The GST-FLASH-DRD fusion protein comprises a forward primer as described in SEQ ID NO: 1 (5'-CGC GGA TCC GAT AAG AGT AAA CTA ACT C-3 ') and a reverse primer as described in SEQ ID NO: 2 (5'-CCG CTC GAG). TTA TTC ACA GGA GCC AGG AGA-3 ') was used to amplify by PCR method. PCR conditions are as follows. After pretreatment at 94 ° C. for 5 minutes, it was repeated for 30 seconds at 94 ° C., 30 seconds at 54 ° C., and 90 seconds at 72 ° C., and post-treatment at 72 ° C. for 5 minutes. The PCR amplification gene was then inserted into Bam H I and Xho I sites of the pGEX4T-3 vector (Amersham-Phramacid Biotech, USA). The expression vector was transformed into E. coli BL21 (DE3) and cultured to induce the expression of GST-FLASH-DRD with 0.2 mM IPTG. The cells were then harvested and disrupted in buffer (50 mM Tris-HCl, pH 7.4), 1 mM DTT, 0.5 mM EDTA, 10% glycerol). The supernatant was then separated using fusion protein using glutathione-Sepharose 4B.

< Example  1-2> bio panning biopanning )

The target molecule, GST-FLASH-DRD, was diluted to a concentration of 100 μg / ml using a buffer (0.1M NaHCO ₃ (pH 8.6)) and dispensed 100 μl into a 96-well plate to coat the plate bottom surface. Another well of the plate was added a blocking buffer containing BSA (0.1 M NaHCO ₃ (pH 8.6), 5 mg / ml BSA, 0.02% NaN ₃ ) and the plate was stirred to ensure that the surface of the well bottom was completely wet. After that, it was left at 4 ° C. for one night. Thereafter, the solution contained in the 96-well plate was removed, and the plate was inverted to remove the remaining solution using a paper towel. 200 μl of blocking buffer was added to the well coated with the target protein and the well containing the blocking buffer, and left at 4 ° C. for 1 hour. The blocking buffer was then removed and washed with 200 [mu] l of TBST (TBS + 0.5% [v / v] Tween-20). The procedure was washed six times repeatedly. Dilute the phage library (PH.D.-12 ^TM Phage Display Peptide Libraray Kit, New England BioLabs, UK) to 4 × 10 ¹⁰ with 100 μl of TBST buffer and place it in a well buffered blocking buffer containing BSA. After the addition and stirring at room temperature for 30 minutes, the phage (Phase) was transferred to the well to which GST-FLASH-DRD was fixed, and stirred for 30 minutes at room temperature. Then, unbound phages were removed from the 96-well plate and washed 12 times with 200 μl of TBST buffer, followed by elution buffer (0.2 M Glycine-HCl (pH 2.2), 1 mg / ml BSA). After adding 100 μl, the cells were left within 10 minutes to elute phages with high affinity from the wells. The solution was transferred to a 1.5 mL tube and 15 μl of 1M Tris-HCl (pH 9.1) was added to neutralize the eluted acidified phage solution.

The resulting phage was amplified by the following procedure. Elution buffer (50 µl) was added to 20 ml of LB (10 g bacto-tryptone 10 g, bacto-yeast extract 5 g NaCl 5 g / 1 L) culture with 200 µl of host cell ER2537 culture incubated overnight, and 250 rpm at 37 ° C. The mixture was incubated for 4.5 hours with stirring. This was followed by centrifugation at 10,000 rpm for 10 minutes to allow the cell mass to precipitate down the tube, and the supernatant transferred to a new tube. The solution thus obtained was centrifuged at the same rate for 5 minutes and only 80% of the supernatant was transferred to a new tube. Thereafter, 1/6 volume of PEG / NaCl (20% (w / v) polyethylene glycol-8000, 2.5 M NaCl) was added and stored at 4 ° C. overnight. The next day, centrifugation at 10,000 rpm for 15 minutes at a temperature of 4 ° C. causes the phage to settle down the tube as a white mass. The supernatant was removed and centrifuged for another minute to remove the supernatant completely. The pellet was re-dissolved in 1 ml TBS buffer and centrifuged at 10,000 rpm for 5 minutes at a temperature of 4 ° C. to make pellets of ER2537 remaining unremoved. After that, the supernatant was transferred to a new tube, and 1/6 volume of PEG / NaCl solution was added and left on ice for about 30 minutes to 1 hour. Again centrifugation at 10,000 rpm for 10 minutes at a temperature of 4 ℃ to carefully remove only the supernatant and dissolve the phage mass precipitated under the tube in 200 μl TBS buffer. The amplified phage was diluted to various concentrations, and titrated to calculate the appropriate concentration.

< Example  1-3> M13  Phage Titer  Measure( titering )

4 μl of 200 mg / ml IPTG and 16 μl of 50 mg / ml X-gal were diluted with 30 μl of dH ₂ O. Then, 50 μl was added to the agar plate, and then applied to the plate surface using a sterile glass spreader. For titer, disperse 200 μl of ER2537 strain incubated with OD value of 0.3 to 0.4, and add 10 μl of LB medium containing phage diluted 1/10 sequentially to determine the appropriate concentration. After infection for about 5 minutes at 3 ml of agarose top (agarose top) solution was shaken. After the agarose top (agarose top) is quickly poured into the agar plate (agar plate) to spread evenly throughout, leave at room temperature for 5 minutes and incubate overnight at 37 ℃ incubator. The next day, the appropriate concentration of the amplified phage was counted by counting the number of blue plaques, and 100 μl TBS buffer solution containing about 2 × 10 ¹¹ phage was used for the next biopanning process. It was.

Thereafter, the process was returned to Example 1-2 and performed three times in the same manner. The phage selected by performing three times is used as a phage to prepare DNA for sequencing by measuring titration. The results of each process are shown in Table 1 below.

Bio panning process INPUT OUTPUT Yield 1 time 1 × 10 ¹¹ pfu / ml 5.1 × 10 ⁶ pfu / ml One Episode 2 1 × 10 ¹¹ pfu / ml 1.4 × 10 ⁷ pfu / ml 2.7 3rd time 1 × 10 ¹¹ pfu / ml 4.4 × 10 ⁷ pfu / ml 8.6

< Example  2> FLASH - DRD Peptide selection showing binding to

< Example  2-1> selected M13  Phage DNA Purification and sequence identification

Dilute 10 μl of the phage elution solution after three biopanning processes to 1/10 ⁴ , 1/10 ⁵ , 1/10 ⁶ , mix with the dissolved agarose top and mix with IPTG. X-gal was poured onto agar plate (agar plate) and incubated overnight. The next day, take 20-30 independent plaques from a plate with less than 100 plaques, dilute the overnight ER2537 strain in 1/100 of LB medium, and dilute the dilution in a 1 ml tube. After each aliquot, each plaque was added to 1 ml of diluted ER2537, incubated for 4.5 hours with shaking at 250 rpm at 37 ° C, followed by centrifugation at 10,000 rpm for 30 seconds, taking ER2537 and plaques to remove the agar from the tube. It was precipitated and only 90% of the solution from the top of the supernatant was transferred to a new 1.5 ml tube. 500 μl of this culture was used for purification of phage DNA for sequencing, and the rest was mixed with the same amount of sterile glycerol and stored at -70 ° C.

200 μl of PEG / NaCl was added to 500 μl of the phage-containing solution, mixed with shaking, left at room temperature for 10 minutes, centrifuged at 10,000 rpm for 10 minutes, and only the solution was carefully removed. Then, 100 μl of iodine buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (Ethylendiamine Tetraacetic acid), 4 M NaI and 250 μl of ethanol were mixed, left at room temperature for 10 minutes, and then at a temperature of 4 ° C. The solution was removed by centrifugation at 10,000 rpm for 10 minutes, and 100 µl of 70% ethanol was added thereto, followed by centrifugation at 10,000 rpm for 5 minutes at 4 ° C. to remove only the solution, and then dried to completely remove the solution in the tube. A single strand of phage DNA was prepared by melting white pellets with tea distilled water.

PCR was performed using a single-stranded phage DNA as a template and using the ABI PRISMTM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, USA) and the -96 pIII sequence primer included in the phage library kit. Was performed. PCR conditions were 5 minutes at 98 ° C., 10 seconds at 98 ° C., 5 seconds at 50 ° C., and 4 minutes at 60 ° C., and amplified DNA was repeated 25 times. The composition of the PCR solution was 4.0 μl of Terminator Ready Reaction Mix, 3 μl of single-stranded phage DNA (30-100 ng), -96gIII base primer (5'-GTA TGG GAT TTT GCT AAA CAA-3) 3.2 pmole and total volume 20 μl. The DNA amplified in the PCR process was precipitated by adding 2 times volume of 100% ethanol and 1/10 volume of 3 M NaOAc, and then heated at 100 ° C. for 3 minutes. DNA obtained through the above procedure was identified by ABI 373 automatic DNA sequencer (Applied Biosystems, USA). The above 20 peptide sequences were obtained (Table 2 and SEQ ID NOs: 4 to 13). As a result of grouping sequences with similar motifs, Group I had the HIHXH motif, Group II had the ASS motif, and Group III did not find similar motifs. In the subsequent process, 10 phage clones of group I were cloned with the phage DNA having the KSLSB HD HIHHH sequence, and the sequence was named "peptide # 1".

order frequency SEQ ID NO: Group I K SLSR HD HIHH H KSLS R HP HIHH H K HIHH SYYRAPN KSLS R PP HIH R H Consensus sequence: KSLSR XX HIHHH   10 2 1 1   4 5 6 7 Group II TMYSQLSF AAS S SHAHPKNTS AAS Consensus sequence: AAS   1 1   8 9 Group III TFTHHRHYPKVVHNHWPYARG SGAGPLQVNRSLLSHHKMHSH PRLTSP   1 1 1 1   10 11 12 13

< Example  2-2> selected M13 Phage  Amplification

Take 200 μl of the ER2537 culture incubated overnight, transfer to a flask containing 20 mL LB medium, inoculate 20 μl in selected M13 phage stock, and incubate for 4.5 h in a 37 ° C. shaking incubator. Incubated. The culture solution was then transferred to a new tube, followed by centrifugation at 10,000 rpm for 10 minutes to precipitate ER2537 under the tube, and the supernatant transferred to a new tube. The solution thus obtained was centrifuged at the same rate for 5 minutes, only 80% of the supernatant was transferred to a new tube, and 1/6 volume of PEG / NaCl solution was added and stored overnight at 4 ° C. The next day, centrifugation at 10,000 rpm for 15 minutes at a temperature of 4 ° C causes the phage to settle down the tube as a white mass. The supernatant is removed and centrifuged for another minute to completely remove the supernatant. The phage mass was re-dissolved in 1 ml of TBS buffer and centrifuged for 5 minutes at 10,000 rpm at a temperature of 4 ° C to precipitate the remaining cell debris under the tube, and then the solution was transferred to a new tube containing 1/6 volume of Add PEG / NaCl solution and leave on ice for 30 minutes to 1 hour. Centrifuge again at 10,000 rpm for 10 minutes at a temperature of 4 ° C to carefully remove only the supernatant and dissolve the phage mass deposited under the tube with 200 µl of TBS buffer. The amplified phage was diluted to various concentrations and titer measurements were performed in the same manner as in Example 1-3.

< Example  3> with selected peptides FLASH - DRD Investigate in conjunction with

< Example  3-1> Selected Peptide Synthesis

Peptides were synthesized based on the most frequent peptide sequences among several sequences obtained through phage display. That is, KSLSRHDHIHHH (SEQ ID NO: 4), which is a peptide sequence with a high frequency of appearance (hereinafter referred to as "peptide # 1"), was ordered to Peptron ^TM ( www.peptron.com ) in the form of binding biotin to the N-terminus. Synthesized.

< Example  3-2> SPR ( Surface Plasmon Resonance : BIAcore  3000) analysis

On the CM5 sensor chip, GST was immobilized using an amide bond as a target molecule, FLASH-DRD, and a background molecule. Activated with 100 μl of NHS / EDC (amin coupling kit, pharmacia biosensor AB, #BR -1000-50) on the surface of the sensor chip protruding from the dextranne attached to the carboxyl group (-COOH) Thereafter, FLASH-DRD and GST were immobilized by diluting with HBS solution. Then, synthesized peptide # 1 was injected 1 mM, 500 μM, 250 μM, 100 μM and 50 μM, respectively, to determine whether binding was increased. The control peptide was 12 mer and peptides having different sequences were injected with 1 mM and 500 μM to compare the binding affinity with peptide # 1.

As a result, as the concentration of peptide # 1 increased, the degree of binding between FLASH-DRD and peptide # 1 was increased (FIG. 3A), but the degree of binding of the control peptide was significantly reduced compared to peptide # 1 (FIG. 3A). 3B). The Kd value of peptide # 1 was calculated to be 5.61 × 10 ⁻⁴ M.

< Example  3-3> In vitro  Combined Assays in in vitro binding assay )

The GST-FLASH-DRD protein was immobilized by treatment with glutathione Sepharose 4B, followed by HBSS buffer (1% NP-40, 100 M PMSF, 2 μg) with caspase-8 labeled [ ³⁵ S] Methionine (Amersham Pharmacia Biotech). / Ml leupeptin, 2 ㎍ / ㎖ aprotinin, 1 v ㎍ / ㎖ pepstatin). Peptide # 1 was added and allowed to stand for 3 hours with stirring at 4 ° C. After that, the bead complex (Bead complex) was washed with HBSS buffer, the bound protein was confirmed by performing the SDS-PAGE.

As a result, it was confirmed that peptide # 1 binds to the DRD domain of FLASH (FIG. 4). Peptide # 1 binds to the DRD domain of FLASH and appears to interfere with the binding of FLASH to caspase 8.

< Example  4> Peptide # 1 Expression Animal cell line  Produce

< Example  4-1> Peptide Expression Vector Preparation

Using a single strand of M13 DNA obtained from the selected phage as a template, a primer for amplifying a random portion of pIII of the DNA was prepared. Ie, SEQ ID NO: 14 for cloning Xho Forward primer with I restriction enzyme site (5'-TCT CTC GAG GTG GTA CCT TTC TAT TCT CAC TCT-3 ') and reverse primer (5'-ATT with Sal I restriction enzyme site) GTC GAC GGC CGA ACC TCC ACC-3 ') (Figure 5A) was used, PCR reaction conditions are as follows. 10 μl of 10 × Mg ⁺⁺ free buffer, MgCl ₂ (25 mM) final concentration 2 mM, forward primer (25 μM) as set out in SEQ ID NO: 14 0.5 μM, reverse primer as set out in SEQ ID NO: 15 μM) 0.5 μM, dNTP mixed solution (10 mM each) 250 μM, M13 DNA (10 ng / μL) 20 ng, and Taq polymerase 1 U (Promega, USA) in a PCR tube Set to μl. 94 ° C. 5 minutes and 94 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. 1 minute were performed, followed by 72 ° C. and 7 minutes after 30 repetitions. After the PCR, the sample was transferred to a new tube, 1/10 volume of 3 M NaOAc and 2 times volume of 100% ethanol were added and left at -20 ° C for 30 minutes. The supernatant was removed after centrifugation at 4 ° C., 13,000 rpm for 30 minutes. Then, 100 μl of 70% ethanol was added and the supernatant was removed after centrifugation at 13,000 rpm for 5 minutes. The PCR DNA was dissolved in 25 μl of dH ₂ O.

< Example  4-2> restriction enzyme reaction and DNA  extraction

Restriction Xho on PCR-Amplified M13 DNA and pEGFP-C3, respectively 2 μl of I 10 U (Roche, USA) and 2 μl of Sal I 10U (Roche, USA) and 10X buffer H were added and the remainder was filled with tertiary sterile distilled water to adjust the total volume to 20 μl. The reaction was carried out at 37 ° C. for 2 hours, and cleavage was confirmed using 1% or 2% agarose gel (1 × TAE). Both the M13 DNA and pEGFP-C3 digested with restriction enzymes were loaded onto a 1% or 2% agarose gel, and the band (87bp) of the random portion of the M13 DNA in the gel was digested. pEGFP-C3 was extracted from each DNA using DNA elution kit (Viogene # EG1001). The extracted DNA was quantified by loading on 1% or 2% agarose gel.

< Example  4-3> Ligation Ligation )

Ligation was performed with the molar ratios of the vector: inserted gene being 1:50 and 1:80. Add 1 μl of 10 × ligation buffer and T4 ligase (1 U / μl) to M13 DNA digested with restriction enzymes and pEGFP-C3, and total volume to 10 μl with tertiary sterile distilled water. The reaction was carried out for 16 hours or more under running water at 16 ° C. (FIG. 5B). The vector obtained by ligation was named pEGFP-SE # 1, and the expression vector in which the gene encoding peptide # 1 was inserted backwards into the vector was named pEGFP- # 1 Reverse.

< Example  4-4> Transformation

E. coli DH5α was inoculated in LB medium (5 ml) and cultured in a 37 ° C. shaking incubator for at least 12 hours. Inoculate 1/100 of colon DH5α in fresh 5 ml LB medium and incubate for 1 hour in a shaking incubator at 37 ° C. to make the OD600 0.5 to 0.6. 1 ml of strain was transferred to the tube and centrifuged at 4,000 rpm for 5 minutes to remove supernatant. 1 ml of cooled 100 mM CaCl ₂ was added to the pellet, and left on ice for 20 minutes. Centrifuged at 4 ° C., 4,000 rpm for 5 minutes and the supernatant was removed. Ligation mixture with 150 μl of cooled 100 mM CaCl ₂ was added, mixed, and left to stand on ice for 30 minutes. Thermal shock was given at 42 ° C. for 1 minute 30 seconds. After standing on ice for 2 minutes, 600 µl of cooled LB medium was immediately added to recover damaged cells. After incubation for 1 hour in a 37 ℃ shaking incubator and plated on 100 L kanamycin (30 ㎍ / ㎖) containing LB plate was incubated for 12-16 hours at 37 ℃, colony (colony) was confirmed.

< Example  4-5> DNA  Extract and Insert Gene Identification

White colonies were harvested and inoculated in 5 ml LB medium containing 30 μg / ml of kanamycin and incubated for 16 hours in a 37 ° C. shake incubator. The cultured E. coli was centrifuged at 1,000 rpm for 10 minutes to remove supernatant. 100 μl of Solution I (50 mM glucose, 25 mM Tris-Cl (pH 8), 10 mM EDTA (pH 8)) was added to the pellet, mixed well until the pellet was completely loosened, and left at room temperature for 5 minutes. 200 μl of solution II (0.2 N NaOH, 1% SDS) was added and shaken up and down, followed by mixing for 5 minutes at room temperature and 5 minutes on ice. 150 µl of solution III (60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of dH ₂ O) was added, shaken up and down, and then placed on ice for 10 minutes. After centrifugation at 4 ° C., 13,000 rpm for 5 minutes, the supernatant was transferred to a new tube. Phenol: chloroform: isoamylalcohol (25: 24: 1) was mixed in the same volume as the sample, centrifuged at 4 ° C and 13,000 rpm for 10 minutes, and the supernatant was transferred to a new tube. 100% ethanol was added at twice the volume of the sample, and stored at −20 ° C. for at least 15 minutes to precipitate DNA. After centrifugation at 4 ° C., 13,000 rpm for 15 minutes, the supernatant was removed. 500 μl of 70% ethanol was added, centrifuged at 4 ° C., 13,000 rpm for 5 minutes, and then the supernatant was completely removed. Dry the DNA pellet at room temperature, dissolve in 31 μl of tertiary sterile distilled water, mix 1 μl DNA, 4 μl of tertiary sterile distilled water, and 1 μl of 6x BPB to mix 1% agarose gel (1 × TAE). Was electrophoresed at 100V for 30 minutes to confirm the size and amount of DNA isolated.

The colonies do not contain the insertion gene, and the insertion gene is reversed (pEGFP- # 1Reverse), so the restriction enzymes Xho I and Sal DNA sequencing confirmed that the insertion gene was inserted into the forward direction (pEGFP-SE # 1). At this time, the primer (5'-CTC TCG GCA TGG ACG AGC-3 ') as set forth in SEQ ID NO: 16 was used to confirm the nucleotide sequence of the insertion gene into the pEGFP-C3 vector.

< Example  4-6> Lipofertamine Using In animal cell lines  Transformation

Hela cells were aliquoted at 10 ⁶ cells / ml and incubated for 24 hours in a 37 ° C. CO ₂ incubator and transformed when the cells grew 50-60%. For transformation using a 35 mm dish, 66 μl of serum free media to which 4 μl of lipofectamine (Invitrogen, USA) was added and 70 μl of serum-free medium to which each DNA mixture was added were mixed. The reaction was carried out for 30 minutes at. After addition of 560 μl serum free medium, 700 μl of the mixture was added to a 35 mm dish. This was followed by incubation for 4.5 hours in a CO ₂ incubator. After incubation, 1 ml of DMEM medium containing 10% FBS was added and then treated with 30 ng / ml TNF-α, an apoptosis inducer, for 24 hours. When a 60 mm dish was used, 294 μl of serum-free medium to which 6 μl of lipofectamine was added and 300 μl of serum-free medium to which respective DNA (5 μg) mixtures were mixed were reacted at room temperature for 30 minutes. 2.4 ml of the mixture and serum-free medium were further added, followed by incubation for 4.5 hours, and medium exchanged with 3 ml of DMEM medium.

< Example  5> In animal cell lines  For peptide # 1 Aptamer In apoptosis  Investigate impact

< Example  5-1> Hoechst  33258 through dyeing Apoptosis  Research

6 wells were coated with cupslip with 0.2% gelatin, then Hela cells were dispensed at 2 × 10 ⁵ cell levels and after 24 hours pEGFP-C3 and pEGFP-SE # 1 1 Μg was transformed. After 24 hours, TNF-α was treated with 30 ng / ml and cyclohexamide 1 ㎍ / ml, and the degree of apoptosis according to the time of day was observed by nuclear shape change through hoechst staining and cell shape change by GFP expression. Hoechst staining process is as follows. Add 1 ml of PBS and 5 µl of Hoechst 33258 (10 mg / ml) to the 35 mm dish, and add the grown coverslips to the 35 mm dish containing the dyeing solution using tweezers. After leaving at room temperature for 2 minutes, the dyeing solution is removed. After washing with 1 ml of PBS, UV and filter were adjusted with a fluorescence microscope (Zeiss Axioplan2) to observe at a magnification of 400 times and photographed (Zeiss MC100).

In addition, in order to confirm that the expression vectors are properly expressed in animal cells, pEGFP-c3 vector and pEGFP-SE # 1 vector were transformed into 293T cells having high transformation efficiency and Western blot (Sambrook, J et al., Molecular Cloning, 2nd ed, Cold Spring Harbor Laboratory Press, 1989) was used to analyze the expression level, anti-GFP antibody (Santa Cruz biotechnology, USA) was used.

As a result, when the pEGF-C3 vector was transformed and treated with TNF-α for 24 hours, the apoptosis was suppressed when the pEGFP-SE1 # vector was transformed and treated with TNF-α (FIG. 6A-D). Red arrows indicate apoptotic cells among the cells expressing GFP and white arrows indicate cells that survived among the cells expressing GFP. The inventors confirmed that Hela cells transformed with pEGFP-SE # 1 inhibited TNF-α induced apoptosis by 30% than when transformed with pEGFP-C3 vector (FIG. 6E). In addition, it was confirmed that each expression vector is normally expressed in an animal cell line (FIG. 6F).

< Example  5-2> FACS  Through analysis Apoptosis  Research

Hela cells were dispensed into 60 mm dishes at 10 ⁶ cells / ml, and then transformed into pEGFP-C3 or pEGFP-SE # 1 24 hours later, and after 24 hours, 30 ng / ml TNF-α and cyclohexamide 1 Cells were harvested 12 h after treatment with μg / ml. The collected cells were allowed to penetrate the cell membrane by leaving 300 μl of PBS and 700 μl of 100% ethanol at 4 ° C. for 30 minutes. Thereafter, cells were collected by centrifugation at 10,000 rpm for 1 minute, the supernatant was removed, and 10 μg / ml of Propidium Iodide (PI) and 5 μg / ml of RNase A were added thereto, followed by reaction at 37 ° C. for 30 minutes, followed by 12,000 rpm. After centrifugation, the supernatant was removed, and cells were dissolved in 400 μl of PBS added with 10% FBS and irradiated with FACStarplus (Becton-Dickinson). The procedure was repeated twice.

As a result, it was found that the transformation of the pEGFP-SE # 1 vector into the Hela cell line inhibited apoptosis more than the transformation of the pEGFP-C3 vector into the Hela cell line (Table 3). As can be seen in Figure 7, it can be seen that apoptosis is inhibited only in GFP ⁺ cells expressing GFP-peptide and there is no effect on GFP ^- cells in which peptide is not expressed.

Experiment 1 (% of apoptotic cells) Experiment 2 (% of apoptotic cells) No processing / pEGFP-C3 1.26 5.51 TNF-α / pEGFP-C3 15.31 37.43 TNF-α / pEGFP-SE # 1 8.8 27.74

< Example  6> peptide  #One Aptamers caspase -8 active and Caspase Investigate impact on -3 cleavage

< Example  6-1> active Caspase -8 and Caspase -3 split measurements

pEGFP-C3 vector and pEGFP-SE # 1 vector were transformed into Hela cell line in the same manner as in Example 4-6, and after 24 hours, the cells were treated with 30 ng / ml TNF-α and 1 μl / ml cyclohexamide. Anti-caspase-8 antibodies (Chung, C.QW., et al ., Neurobiol . Dis . 14: 557-566, 2003) and anti-caspase-3 antibodies (Santa Cruz biotechnology, USA) were used to perform Western blot analysis by the method of Example 5-1.

As a result, caspase-8 was activated when TNF-α was treated in the Hala cell line transformed with pEGFP-C3 vector, but caspase- was treated when TNF-α was treated with Hala cell line transformed with pEGFP-SE # 1 vector. It was confirmed that the activity of 8, peptide aptamer # 1 inhibits the binding of FLASH-DRD and caspase-8. In addition, in the expression of caspase-3, the Hela cell line transformed with pEGFP-SE # 1 inhibits the expression of caspage-3 as compared with the Hela cell line transformed with pEGFp-C3 (FIG. 8A).

< Example  6-2> active caspase Immunochemical analysis for -8 analysis )

6 wells were coated with coverslip with 0.2% gelatin, then Hela cells were dispensed at 2 × 10 ⁵ cell levels and after 24 hours pEGFP-C3 and pEGFP-SE # 1 1 Μg was transformed. After 24 hours, the cells were treated with 30 ng / ml TNF-α and 1 ㎍ / ml of cyclohexamide, and the changes in the cell morphology of GFP expression and antibody binding to activated caspase-8 were observed.

As a result. In Hala cells transformed with pEGFP-C3 vector, caspase-8 was activated, but in Hala cells transformed with pEGFP-SE # 1 cells, caspase-8 activation was reduced (FIG. 8B).

< Example  6-3> TNF -α and etoposide  origin Apoptosis  Research

pNFP-C3, pEGFP-SE # 1, or pEGFP- # 1 reverse vector was transfected into Hela cell lines in the same manner as in Example 4-6, and treated with TNF-α 30 ng / ml or etoposide 50 μM 24 hours later. The degree of apoptosis of cells was examined at each time period.

As a result, it was confirmed that apoptosis was suppressed in the Hela cell line transformed with pEGFP-SE # 1 than the Hela cell line transformed with the pEGFP-C3 vector (FIG. 8C). Etoposide is a Topoisomerase II inhibitor that causes DNA damage and releases mitochondrial cytochrome C, causing apoptosis. When etoposide was treated, pEGFP-SE # 1 vector was transformed into Hela cells, and thus did not inhibit apoptosis because there was no change unlike TNF-α treatment (FIG. 8D). This suggests that peptide # 1 aptamer specifically inhibits only TNF-α-derived apoptosis. pEGF- # 1 Reverse vector does not affect both cases treated with TNF-α or etoposide.

< Example  7> Peptide # 1 Variants On apoptosis  Investigate impact

< Example  7-1> Peptide # 1 Variant  making

The inventors prepared variants based on peptide # 1 and investigated the effects on apoptosis. The sequence of the variant is shown in Table 4 below, and the preparation method was performed as in Example 3-1.

Peptide # 1 Variant Sequence SEQ ID NO: K S L S R M I I F I I 17 K S L S R H D H T H H H 18 E S L S R H D H I H H H H 19 K S L S R H D L F I I I 20 K S L I R H D H I H H H H 21

< Example  7-2> Peptide # 1 variant Apoptosis  Impact investigation

The inventors transformed the pEGFP vector in which the PEGFP-c3 empty vector and the peptide # 1 and its variants (SEQ ID NOs: 17 to 21) were inserted into the HeLa cell line in the same manner as in Example 4-6. TNF-α (30 ng / ml) was treated for 18 hours after 24 hours after transformation, and cell death was measured to the extent of GFP expression and nuclear condensation.

As a result, the variant of SEQ ID NO: 17 was not well expressed and the degree of cell death could not be measured. 21 of the other variants (SEQ ID NO: 18 to 21) was confirmed to inhibit apoptosis like peptide # 1 (Fig. 9).

< Example  8> FLASH of Antisense  Nucleotide Production

Anticent nucleotides were made complementary based on mRNA of human FLASH. An antisense nucleotide (5'-TAA GTT GCT GAA T-3 ') described in SEQ ID NO: 22 was produced by Bionnia (Korea). The sequence is located in the 6138-6155 bp downstream portion of the translation initiation site in the coding region. A control sequence described in SEQ ID NO: 23 (5′-ATG CTG CAG CAT GAT CTA-3 ′) was prepared and used as a control. Based on the sequences were synthesized chemically.

< Example  9> FLASH of Antisense  Nucleotide Apoptosis  Impact investigation

Jurkat cell lines were dispensed at 3 × 10 ⁶ cells / well and then transformed into cell lines by the method of Example 4-6, treated with 5 μM FLASH antisense nucleotides or 5 μM control antisense nucleotides. We used TNF-α (30 ng / ml) / cyclohexamide (10 μg / ml) or anti-Fas antibody (60 ng) to induce apoptosis by receptor signal 48 hours after the transformation. / Ml) (Santa Cruz Biotechnology, USA) to examine the degree of apoptosis by time zone in the same manner as in Example 6-3. In addition, A23187 (0.3 μM), Staurosporine (0.2 μM), and etoposide (60 μM), which induce apoptosis by non-receptor signals, were treated to investigate the degree of apoptosis by time zone.

As a result, it was confirmed that the FLASH antisense nucleotides inhibited Fas-derived apoptosis as well as TNF-α-derived apoptosis (Figs. 10A and 10B). In contrast, FLASH antisense nucleotides did not inhibit apoptosis by non-soluble signals of A23187, Staurosporine, etoposide (FIGS. 10C-10E).

We performed RT-PCR to investigate the effect of FLASH antisense nucleotides on transcriptional expression of FLASH. After treating the apoptosis inducing substance, the total RNA was extracted, and then cDNA was synthesized by 5 min at 25 ° C, 60 min at 42 ° C, 15 min at 70 ° C. Primers were performed using the forward primer set forth in SEQ ID NO: 1 and the reverse primer set forth in SEQ ID NO: 2. PCR conditions are as follows. After 1 minute of pretreatment at 94 ° C., it was repeated 30 times at 94 ° C., 30 seconds at 55 ° C., 2 minutes at 72 ° C., and post-treatment at 72 ° C. for 5 minutes.

As a result, it was confirmed that FLASH antisense nucleotides inhibited transcriptional expression of FLASH in cells (FIG. 10F).

The method for inhibiting cell apoptosis by inhibiting the activity of FLASH (FLICE-associated huge protein) of the present invention is FLASH comprising an antisense nucleotide complementary to FLASH peptide aptamer or FLASH mRNA as an active ingredient. Through activity inhibitors. The peptide aptamer of FLASH of the present invention inhibits TNF-α-derived apoptosis, and FLASH antisense nucleotides effectively inhibit TNF-α-derived and Fas-derived apoptosis, resulting in excessive stroke and neurological diseases. It can be useful for the development of therapeutics

Attach sequence list electronic file

Claims (13)

delete delete delete Peptide aptamer set forth in SEQ ID NO: 21 that specifically binds FLASH (FLICE-associated huge protein). delete delete 5. The peptide aptamer of claim 4, wherein the aptamer inhibits TNF-α induced apoptosis. delete delete delete A therapeutic agent for a disease associated with cell death selected from the group consisting of stroke, Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease, comprising the peptide aptamer of claim 4 as an active ingredient. delete delete

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