KR100818752B1 - 1 composition for treating an ischemic disease comprising a human faf1 protein inhibitor as an active ingredient - Google Patents

Abstract

A human FAF1 protein inhibitor is provided to inhibit expression or function of an FAF1 protein effectively and inhibit ischemic cell death effectively by acting on a caspase non-dependent cell death passage during the ischemic cell death, and generation of reactive oxygen species, activation of PARP-1, and a nucleus movement passage of AIF, thereby being used as a pharmaceutical composition for treating or preventing ischemic diseases. A composition for treating or preventing ischemic diseases comprises a human FAF1(Fas-associated factor 1) inhibitor as an effective ingredient, which is selected from the group consisting of a double stranded siRNA consisting of a forward single stranded siRNA having a sequence of SEQ ID : NO. 3 and a reverse single stranded siRNA having a sequence of SEQ ID : NO. 4, a double stranded siRNA consisting of a forward single stranded siRNA having a sequence of SEQ ID : NO. 5 and a reverse single stranded siRNA having a sequence of SEQ ID : NO. 6, and a protein having an amino acid sequence of SEQ ID : NO. 7, wherein the ischemic diseases is cerebral ischemia, cardiac ischemia, diabetic cardiovascular diseases, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, and ischemic acute renal failure mediated by ischemic cell death.

Description

COMPOSITION FOR TREATING AN ISCHEMIC DISEASE COMPRISING A HUMAN FAF1 PROTEIN INHIBITOR AS AN ACTIVE INGREDIENT}

FIG. 1 induces chemical hypoxia (CH) for 90 minutes in H9c2 cells transiently transformed with MOCK (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA, respectively, followed by DAPI (4 ′, 6- diamidino-2-phenylindole, dihydrochloride) analysis was performed to evaluate the percentage of dead cells,

FIG. 2 shows chemical ischemia for 90 minutes in H9c2 cells transiently transformed with MOCK and pFlag-hFAF1 plasmid DNA, respectively, followed by XTT (sodium 3 '-[1- (phenylaminocarbonyl) -3,4-tetrazolium]- cell viability was evaluated by bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (ABS) assay.

3 shows Western blot analysis using HeLa cells transformed with scRNA (scrambled RNA) and siRNA-hFAF1, and anti-FAF1 antibody and anti-β-tubulin antibody. Confirm the expression level of the protein,

4 shows Western blot analysis using H9c2 cells transformed with scRNA and siRNA-rFAF1, and anti-FAF1 antibody and anti-β-tubulin antibody to confirm expression levels of FAF1 protein.

FIG. 5 shows the induction of chemical ischemia for 90 minutes in HeLa cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-hFAF1, respectively, followed by DAPI analysis to evaluate the percentage of dead cells.

FIG. 6 shows that induction of chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1, respectively, followed by DAPI analysis to evaluate the percentage of dead cells.

7 is a chemical ischemia induced in H9c2 cells transformed with MOCK plasmid DNA and siRNA-rFAF1, and then observed the degree of cell death using FACS (fluorescence activated cell sorter, BD),

8 shows the degree of cell necrosis by measuring propidium iodide (PI) -positive cell number after FACS analysis.

9 shows chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1 with GFP-Casease-8-DED, respectively, followed by immunoimmunochemical analysis (immunocytochemical). shows the results of observing the formation of death effector filament (DEF) by performing an analysis,

FIG. 10 shows that H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1, respectively, were treated or untreated with the caspase inhibitor z-VAD-fmk and induced chemical ischemia for 90 minutes An analysis was performed to evaluate the percentage of dead cells,

FIG. 11 shows the induction of chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1, respectively, and then evaluating the generation of reactive oxygen species.

Figure 12 induces chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1, respectively, followed by activity of PARP-1 (poly (ADP-ribose) polymerase-1). Evaluated,

FIG. 13 shows chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA and siRNA-rFAF1, respectively, and then fractions of nuclei and mitochondria obtained by collecting cells, and anti-AIF (apoptosis inducing factor). ) Western blot analysis using antibody, anti-PARP-1 antibody and anti-SOD-2 (superoxide dismutase-2) antibody to observe nuclear transfer of AIF,

FIG. 14 shows that induction of chemical ischemia for 90 minutes in H9c2 cells transformed with MOCK and pFlag-hFAF1 plasmid DNA, and pFlag-hFAF1 [366-650], respectively, followed by DAPI analysis to evaluate the percentage of dead cells. Will be

15 is a schematic of pFlag-hFAF1 [366-650] (FAF1-ΔFID.DEDID, DN (dominant negative)) (FID: Fas-interacting domain); and DEDID: DED -DED-interacting domain.

The present invention relates to a composition for treating ischemic disease containing a human FAF1 protein inhibitor as an active ingredient, a method for inhibiting ischemic cell death by inhibiting human FAF1 protein, and a method for searching for candidates related to ischemic cell death.

Ischemia refers to a reduced state of blood supply to body organs, tissues or sites caused by contraction or blockage of blood vessels. After ischemia, even if reperfusion of blood occurs, nerve cells are damaged to cause various sequelae. Such ischemia is often associated with coronary artery disease, cardiovascular disease, angina pectoris, headache or other vascular symptoms. Such ischemia ultimately leads to irreversible damage, ie necrosis of cells and tissues.

Ischemic diseases such as myocardial infarction, arrhythmia, and insufficiency caused by cellular damage and deterioration during ischemia / reperfusion have high prevalence, mortality, and difficulty in cure. See Wang, QD et al., Cardiovasc. Res. 55: 25-37, 2002). Ischemia / reperfusion injury involves various physiological mechanisms such as metabolism, changes in immune response and ionic constant, oxygen free radicals, and therefore, research is being conducted in various fields such as immunomodulators, apoptosis-related substances, and ion channel regulators. Hearse, DJ et al., Mol. Cell. Biochem. 186: 177-184, 1998). To date, there have been active researches on the development of therapeutic agents and surgical procedures with new action points, but the techniques for protecting cardiomyocytes from ischemia / reperfusion have not been commercialized clinically. Therefore, there is a need for development of a prophylactic or therapeutic agent for ischemic heart disease or a cardioprotective agent that can slow the progression of cardiomyocyte damage due to ischemia and alleviate reperfusion injury.

It is also increasingly clear that when ischemia is eliminated by the return of blood flow, the production of reactive oxygen species (ROS) is accelerated, which leads to a much more pronounced decrease in glutathione leading to the development of more serious diseases. have. Similar diseases are observed upon stopping or returning blood flow in the implantation of various organs such as the heart, liver, lungs, pancreas and blood vessels. The disease is also a problem in the incision and removal of organs. Free radicals and reactive free radicals that are believed to cause disease are detected in both the cytoplasmic cells and organelles that make up tissue, especially mitochondria producing ATP, which contributes to the cell's primary energy source. In mitochondria, it has been observed that the respiratory chain is the main source of these reactive molecules and their concentrations rise significantly during ischemia and reperfusion.

Ischemia / reperfusion injury is due to an increase in the production of Ca ²⁺ and reactive oxygen species in the cytoplasm, which is an essential cause of cell death. DNA strand degradation induced by reactive oxygen species is due to the excessive consumption of energy sources NAD ⁺ and ATP during apoptosis or necrosis, resulting in the nuclear enzyme poly (ADP-ribose) polymerase-1. Induces overactivation of (poly (ADP-ribose) polymerase-1, PARP-1). Activation of PARP-1 leads to apoptosis of mitochondrial to nuclei, DNA condensation and fragmentation of the apoptosis inducing factor (AIF), which is independent of NAD ⁺ deficiency and caspase ( See van Wijk, SJ et al., Free Radic. Biol. Med. 39: 81-90, 2005).

In ischemic diseases, apoptosis or cell necrosis is caused by ischemia. Especially, apoptosis after reperfusion is a major cause of tissue damage. Ischemic cell death is caused by cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, cardiomyopathy, and retina. Causes of the development of various ischemic diseases, including ischemia, ischemic colitis and ischemic acute renal failure.

In the case of ischemic disease, cerebral ischemia, depletion of energy source causes depletion of energy source, causing ischemic cell death, excessive activation of cell membrane receptors, glutamic acid outside the cell and calcium accumulation inside the cell. It involves a variety of biochemical changes, such as damaging lipids, proteins and nucleic acids, resulting in damage to brain tissue (Liu, PK, J. Biomed. Sci. 10: 4-13, 2003; Lipton, P , Physiol. Rev. 79: 1431-1568, 1999; and Renolleau, S. et al., Stroke 29: 1454-1460, 1998).

In the case of ischemic heart disease, myocardial infarction, arrhythmia and heart failure, it is reported that lipid membrane activation causes cell membrane damage, pH change and calcium migration, resulting in ischemic cell death (Ferrari, R. Rev. Port). Cardiol. 5: 7-20, 2000; Webster, KA et al., J. Clin.Invest . 104: 239-252, 1999; Katz, AM et al., J. Mol. Cell.Cardiol. 2:11 . -20, 1985; and Vandeplassche, G. et al.Basic Res. Cardiol. 85: 384-391, 1990), and in the case of retinal ischemia, glutamate-mediated retinal cell death is associated with ischemic cell death. Is known (see Napper, GA et al., Vis. Neurosci. 16: 149-158, 1999), and ischemic cell death occurs due to insufficient blood supply to the large intestine, and From the above body fluid when the ischemic colitis in ischemic diseases (literature [Saegesser, F. et al, Pathobiol Annu 9:... 303-337, 1979] Trillion).

In addition, minocycline, a tetracycline-based antibiotic that inhibits ischemic cell death, is known to have cerebral infarction (see Yrjanheikki, J. et al., Proc. Natl. Acad. Sci. USA 96: 13496-13500, 1999), myocardial infarction ( See Scarabelli, TM et al., J. Am. Coll. Cardiol. 43: 865-874 , 2004) and ischemic acute renal failure (Wang, J. et al., J. Biol. Chem. 279: 19948-19954, 2004), and is also effective in the treatment of ischemic diseases, it can be seen that ischemic cell death causes the disease.

On the other hand, the release of CD95L, TRAIL and TNFα, known as death inducing ligand (DIL) during ischemia, increases, and therefore, the regulation of apoptosis process is particularly important in ischemic diseases.

Fas (aka CD95 or Apo1) is one of the well defined apoptosis receptors. Fas and Fas ligands (FasL) play an important role in cell death. When three FasLs are bound to three Fas molecules, an adapter protein called a Fas-associated death domain (FADD) consisting of a death domain (DD) and a death effector domain (DED) forms DD. To the DD of Fas. When the DED of FADD binds to the DED of inactive caspase-8 (aka FLICE or MACH), caspase-8 is activated and activated caspase-8 causes downstream effectors ( effector) activates caspase (see Marsters, SA et al., Curr. Biol. 8: 525-528, 1998).

FAF1 was first reported as a protein that binds to Fas receptor through yeast 2-hybrid system screening (Chu, K. et al., Proc. Natl. Acad. Sci. USA. 92: 11894 -11898, 1995). FAF1 is a protein that binds to the cytoplasmic domain of Fas, but is one of the proteins that binds to Fas without conventional DD (Ryu, SW et al., Biochem. Biophys. Res. Commun. 286: 1027-1032, 2001). Reference). FAF1 is a pro-apoptotic protein that has the ability to increase apoptosis induced by Fas and can cause cell death upon overexpression of FAF1 in BOSC cells without Fas signaling. FAF1 also induces cell death by forming Fas, FADD, caspase-8 and Fas-death inducing signaling complex (Fas-DISC) (Ryu, SW et al, J. Biol. Chem. 278: 24003-24010, 2003). It has also been reported to be involved in receptor-independent cell death pathways, thereby enhancing cell death sensitivity of cancer cells by conventional anticancer agents.

Death effector filament (DEF) is a new cytoskeletal construct that is one of a collection of DEDs, the formation of which causes nonreceptor cell death. FADD, TRADD, caspase-8, caspase-2, caspase-10, etc., including DED or DD, are reported as proteins forming DEF (Perez, D. et al., J. Cell Biol. 141: 1255-1266, 1998; Tsukumo, SI et al., Genes Cells. 4: 541-549, 1999; and Shikama, Y. et al, Biochem. Biophys.Res.Commun . 291: 484 -493, 2002).

In the previous study, we observed increased anticancer sensitivity in cells overexpressing FAF1 and found that FAF1 increased anticancer drug induced cell death as a new member of the DEF aggregate (Park, MY et al., Int. J. Cancer. 115: 412-418, 2005).

Therefore, the present inventors continued to search for a novel protein that acts as a modulator of ischemic cell death activity. As a result, overexpression of FAF1 in cells promotes ischemic cell death, and siRNA of FAF1 or FAF1 by dominant-negative FAF1. By revealing that ischemic cell death is inhibited by expression or function inhibition, it was confirmed that FAF1 is an essential protein in the process of ischemic cell death.

Furthermore, the present inventors found that FAF1 is essential in caspase-independent cell death among several pathways of ischemic cell death, and plays an important role in ischemic cell death through the generation of reactive oxygen species, PARP-1 activation, and nuclear migration pathway of AIF. Confirmed.

Accordingly, an object of the present invention is to provide a composition for treating ischemic disease that can effectively suppress ischemic cell death.

Another object of the present invention is to provide a method for effectively inhibiting ischemic cell death.

Still another object of the present invention is to provide a method for effectively searching for candidate substances related to ischemic cell death.

In accordance with the above object, the present invention provides a composition for the treatment or prevention of ischemic diseases containing a substance that inhibits the expression or function of human FAF1 protein as an active ingredient.

According to the above another object, the present invention provides a method for inhibiting ischemic cell death by inhibiting the expression or function of FAF1 protein in a subject in need of inhibition of ischemic cell death.

In accordance with another object, the present invention provides a method for searching for candidates that inhibit ischemic cell death by using an antibody against the FAF1 gene or the FAF1 protein.

Hereinafter, the present invention will be described in detail.

As used herein, the term 'FAF1 protein' refers to an isolated native polypeptide that plays an essential role in ischemic cell death and enhances ischemic cell death, specifically SEQ ID NO: 1 Polypeptides having amino acid sequences and splicing and allelic variants thereof.

The 'natural polypeptide' is a polypeptide present in all cell types of human or non-human mammal species, including everything with or without starting methionine, as well as purification, chemical synthesis or DNA recombination from natural sources. It includes everything produced by a technique or prepared by a combination of these and / or other methods. In this respect, natural polypeptides obtained from humans as well as other mammalian species, such as pigs, dogs, horses and the like, are also included in the present invention.

The term 'isolated', as used in the context of a protein, polypeptide or nucleic acid, refers to a state in which the protein, polypeptide or nucleic acid is released from at least some of the proteins, polypeptides or nucleic acids bound to the natural environment. .

The composition for treating ischemic disease of the present invention is characterized by containing a substance effectively inhibiting the expression or function of human FAF1 protein as an active ingredient.

Substances that inhibit the expression or function of the human FAF1 protein include antisense RNA, iRNA (interfering RNA) and siRNA (small interfering RNA), which typically knock out the FAF1 gene at the mRNA level, and An RNA expression vector capable of introducing the expression inhibitory RNAs into a cell; Dominant-negative (DN) FAF1 protein; Transcription inhibitors of the FAF1 gene; Translation inhibitors of transcribed FAF1 mRNA; Inhibitors of localization of FAF1 protein to specific locations in cells; And antibodies against FAF1 protein can be used. Of these, siRNA and dominant-negative FAF1 protein, which can inhibit the expression or function of FAF1 protein in specific but effective amount, are preferred.

Small RNA fragments, called siRNAs, are small RNA fragments of 21-25 nucleotides in size that are produced by cleavage of double-stranded RNA by Dicer enzymes specifically for mRNAs having complementary sequences. It can be used to bind and inhibit protein expression. siRNAs are designed to complement regions of genes involved in transcription and thus can inhibit transcription and production of FAF1 protein.

In the present invention, as a substance for inhibiting the expression of human FAF1 protein provides a siRNA consisting of 19 base pairs, preferably a forward single stranded siRNA having a nucleotide sequence of SEQ ID NO: 3 and a reverse single stranded siRNA of SEQ ID NO: 4 Provided are double stranded human FAF1 siRNAs, and a forward single stranded siRNA having the nucleotide sequence of SEQ ID NO: 5 and a reverse single stranded siRNA of SEQ ID NO: 6.

In addition, the present invention provides a dominant-negative FAF1 protein as a substance that inhibits the function of the human FAF1 protein, and preferably, the dominant-negative FAF1 protein FAF1 [366-650] (FAF1) having the amino acid sequence of SEQ ID NO: 7. -ΔFID DEDID) (see FIG. 15). The FAF1 [366-650] comprises amino acids 366 to 650 of the FAF1 protein having an amino acid of SEQ ID NO: 1, and includes a fas-interacting domain (FID) and a DED-binding region (DED-interacting) of FAF1. domain, DEDID) and is encoded by DNA having a nucleotide sequence of SEQ ID NO: 8 (Ryu, SW et al., Biochem. Biophys. Res. Commun. 262: 388-394, 1999; and Ryu, SW et al, J. Biol. Chem. 278: 24003-24010, 2003).

The dominant-negative FAF1 protein includes a functional derivative thereof. The term 'functional derivative' typically refers to a compound that exhibits a biological activity substantially comparable to a natural polypeptide, and for the purposes of the present invention, a functional derivative of the FAF1 protein has biological activity equivalent to that of FAF1 since it inhibits ischemic cell death. This means that the derivative binds to FAF1 and inhibits the physiological activity mediated by FAF1.

The functional derivative of the dominant-negative FAF1 protein according to the invention is preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80% with the native FAF1 protein, preferably human FAF1 protein. Most preferably, amino acid sequence variants having at least about 90% amino acid sequence homology and wherein some of the amino acids of the natural protein have been mutated by substitution, deletion or addition, unless the inherent biological activity is lost . Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids include aliphatic amino acids (Gly, Ala, Pro), hydrophobic or aromatic amino acids (Ile, Leu, Val, Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur-containing amino acids (Cys, Met).

On the other hand, polypeptides having a biological activity substantially equivalent to native FAF1 as fragments of FAF1 protein from several mammalian species form another preferred group of functional derivatives of FAF1 protein. The term “fragment” refers to an amino acid sequence corresponding to a portion of a protein, which has a common element of origin, structure and mechanism of operation within the scope of the present invention and which is naturally occurring, cleaved using a protease or chemically. All cuts are included. In addition, functional derivatives modified to alter the stability, shelf life, solubility, etc. of the native FAF1 protein, or modified to change the relationship with a substance interacting with, for example, FAF1, may also be used as functional derivatives of the FAF1 protein according to the present invention. Another preferred group is formed.

The term 'similarity' or 'homologous' as used in connection with a natural protein or functional derivative thereof refers to a gap to incubate the corresponding candidate sequence identical to the amino acid residues of the natural protein or functional derivative thereof and to obtain the maximum homology ratio if necessary. After the introduction, the ratio of amino acid residues similar to the candidate sequence according to the present invention is indicated. The numerical value of sequence similarity is well defined in the NCBI BLAST 2.0 software (Altschul et al., 1997).

The dominant-negative FAF1 protein, functional derivatives, fragments thereof and all variants thereof of the present invention can be prepared using one of the known organic chemical methods for peptide synthesis. Organic chemical methods of peptide synthesis include coupling the required amino acids by a condensation reaction in homogeneous phase or with the so-called solid phase assistance. The most common methods for condensation reactions include the carbodiimide method, the azide method, mixed anhydride methods and methods using active esters (Gross, E. et al., The peptides Analysis, Synthesis, Biology Vol. 1-3, 1997-1981, Academic Press).

Particularly suitable solid phases for the synthesis of peptides are p-alkoxybenzyl alcohol resins (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-1% divinylbenzene resins) (Wang, J. Am. Chem. Soc ., 95, 132, 1974), the synthesized peptide contains trifluoroacetic acid containing a scavenger such as triisopropyl silane, anisole or ethanedithiol, thioanisole under mild conditions. Can be isolated from the solid phase. Reactive groups that cannot be involved in the condensation reaction are effectively protected by groups that can be removed very easily again by hydrolysis or reduction with acids, bases. For protecting groups that may possibly be used, see Gross, et al., The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, 1979-1987, Academic Press. In addition, peptide sequences synthesized in fragments can be combined with each other to obtain the desired full length sequence.

In addition, in the present invention, a single or polyclonal antibody against FAF1 protein can be used as a substance that inhibits the function of human FAF1 protein. Such antibodies can be readily prepared using known techniques (see James W. Goding, Monoclonal Antibodies: Principles and Practice, Second Ed., Academic Press, 1986), and fragments of antibodies (eg Fab fragments). ), Humanized antibodies and all types of antibodies are included in the antibody scope of the present invention.

Since the composition for treating ischemic disease of the present invention contains a substance effectively inhibiting the expression or function of human FAF1 protein, it can selectively inhibit ischemic cell death activity.

The composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and for this purpose, a pharmaceutically acceptable carrier, in addition to an inhibitor of expression or function of FAF1 protein, which is used as the active ingredient. For example, it may further comprise a water-soluble carrier, excipients or other additives as the case may be. The formulations may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders, etc., for use in gene therapy, etc. It is preferable to formulate it as an injection for intravenous injection, subcutaneous injection, endothelial injection, intramuscular injection and the like.

Specifically, the double-stranded siRNA according to the present invention or a composition comprising the same can be carried out by a known method introduced into target cells, and conventional gene transfer techniques include calcium phosphate method, DEAE-dextran method, electroporation, micropores. Injection, viral, and methods using cationic liposomes (Graham, FL et al., Virol . 52, 456, 1973; McCutchan, JH et al., J. Natl. Cancer Inst . 41, 351, 1968; Chu, G. et al., Nucl.Acids Res. 15, 1311, 1987; Fraley, R. et al., J. Biol. Chem. 255, 10431, 1980; Capecchi, MR et al., Cell , 22, 479, 1980; and Felgner, PL et al., Proc. Nati. Acad. Sci. USA , 84, 7413, 1987). In one embodiment of the present invention, siRNA of the present invention was administered by the Amaxa Nucleofector ^™ electroporation method in combination with the electroporation buffer.

The siRNA of the present invention may be administered in milligram or microgram amounts per subject or sample dose, for example 1 μg to 500 mg / kg, preferably 1 μg to 50 mg / kg, more preferably 100 μg to It may be administered once per week for 1 to 10 weeks in an amount of 5 mg / kg.

In addition, the dominant-negative FAF1 of the present invention can be directly injected or formulated in a solution or micelle form, and the daily dose of the inhibitor or expression of FAF1 protein to be administered to the patient for clinical purposes is 0.05 to 200 mg. / kg body weight, preferably in the range of 0.05 to 150 mg / kg body weight can be used to inhibit the expression of the FAF1 protein, it can be administered once or divided into several times. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and thus the dosage should be determined in any aspect of the invention. It does not limit the scope of.

The present invention also provides a method for inhibiting ischemic cell death by inhibiting the expression of FAF1 protein in a subject in need of inhibition of ischemic cell death, wherein the subject may be a mammal including a human.

In the present invention, a method of inhibiting the expression or function of the FAF1 protein includes a method of targeting the FAF1 gene on DNA by conventional gene targeting; Targeting mRNA of FAF1 using antisense RNA, iRNA and siRNA, and RNA expression vector capable of introducing such expression inhibitory RNAs into cells; Binding a dominant-negative FAF1 protein with FAF1 to inhibit FAF1 mediated physiological activity; How to inhibit transcription of the FAF1 gene; A method of inhibiting translation of transcribed FAF1 mRNA; The antibody injection method or the method of inhibiting the movement of FAF1 protein to a specific position in the cell, and the like, but is not limited thereto.

Since the method of inhibiting expression or function of FAF1 protein of the present invention effectively inhibits intracellular ischemic cell death, ischemic diseases such as cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, cardiomyopathy, retinal ischemia, ischemic colitis and ischemic acute renal failure. It can be used for the treatment or prevention of.

In one embodiment of the invention FAF1 also enhances anticancer agent-induced cell death by increasing the formation of DEF as a new member of DEF (Park, MY et al., Int. J. Cancer. 115: 412-418, Free radical species are generated upon ischemia induction (see Bonne, C. et al., Gen. Pharmacol. 30: 275-280, 1998), and the generation of reactive oxygen species activates PARP-1. (See van Wijk, SJ et al., Free Radic. Biol. Med. 39: 81-90, 2005), and increasing PARP-1 activity releases AIF protein from the mitochondria and transfers it to the nucleus ( Based on the facts, see van Wijk, SJ et al., Free Radic. Biol. Med. 39: 81-90, 2005). Based on the facts, the relationship between FAF1 and the facts using FAF1 overexpression and siRNA of FAF1 In addition, FAF1 was examined via the caspase cell death pathway in ischemic cell death. As a result, it was confirmed that FAF1 plays an important role in ischemic cell death process through the formation of DEF, generation of reactive oxygen species, PARP-1 activation, and nuclear migration pathway of AIF, and is essential in caspase-independent cell death process.

In addition, the present invention provides a method for searching for candidate substances that inhibit ischemic cell death by using an antibody to the FAF1 gene or the FAF1 protein.

Specifically, the search method may search for another core protein that binds to FAF1 protein that regulates ischemic cell death of FAF1 protein upon ischemia induction, or may be a candidate of a therapeutically active substance for ischemic disease that modulates the function of FAF1 protein, or The method may be a method of searching for a candidate substance for controlling ischemic cell death in cells by searching for a candidate substance for controlling cell death or reactive oxygen species caused by FAF1 during ischemia.

In the search method, the candidates are treated with FAF1 protein-expressing cell lines, tissues, and experimental animals, and then the expression level of the FAF1 gene or protein is confirmed using an FAF1 gene or an antibody to the FAF1 protein, or the ability to inhibit ischemic cell death. In this way, it is possible to search for candidate substances that effectively inhibit the expression of FAF1 protein and regulate ischemic cell death.

As a method for assaying the expression level of the FAF1 gene or FAF1 protein and the ability to inhibit cell death that can be used in the search method of the present invention, general biochemical techniques used to confirm the expression of the gene or protein in the art may be used. . For example, methods for confirming the expression of the FAF1 gene include reverse transcriptase polymerase chain reaction (RT-PCR) and various blot analysis using the FAF1 gene or fragments thereof as probes, such as Northern blot analysis. Examples of methods for confirming expression of FAF1 protein include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and sandwich assay (sandwich assay) using antibodies against FAF1 protein. ), Western blots on polyacrylamide gels, immunoblot analysis and immunohistochemical staining.

For example, when screening for inhibitors of FAF1 function in ischemia, the FAF1 protein is overexpressed in cells and induces ischemia to create optimal conditions for increased ischemic cell death, followed by treatment of candidates and their inhibitory activity for ELISA analysis. It can be confirmed by conventional methods such as cell staining analysis.

In addition, the present invention can provide a method for isolating molecules that selectively bind FAF1. The method includes incubating the mixture of FAF1 with the candidate molecule, isolating the complex, and freely removing the FAF1 protein from the isolated complex. Isolation of the complex can be performed by a variety of conventional isolation methods, including, for example, an affinity column immobilized with FAF1 protein, immunoprecipitation using an anti-FAF1 antibody, a yeast two-hybrid system, and the like.

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: Confirmation of FAF1 and Ischemic Cell Death

The following experiments were performed to investigate the effect of FAF1 on ischemic cell death.

(1-1) Preparation of Transgenic Cell Line

Rat cardiomyocyte line H9c2 cells (ATCC, CRL-1446) using DMEM (Dulbecco's modified Eagle's medium, JBI) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100 × solution) After incubation in a 5% CO ₂ incubator at 37 ° C, 1 to 2 × 10 ⁶ H9c2 cells were collected and washed once with PBS, and then reproduced in 100 μl of a speculative electroporation buffer (Amaxa). It was cloudy. 1 μg of pFlag-hFAF1 plasmid DNA (Ryu, SW et al., Biochem. Biophys. Res. Commun. 262: 388-394, 1999; and Ryu, SW et al, J. Biol. Chem. 278: 24003-24010, 2003) and MOCK (pFlag-CMV-5a plasmid DNA, sigma) were mixed, transferred to cuvettes, and transformed cell lines using an Amaxa Nucleofector ^™ device (Amaxa). Prepared.

(1-2) Observation of ischemic cell death

In order to induce chemical hypoxia (CH), the transformed H9c2 cells of (1-1) were incubated for 48 hours at 37 ° C., CO ₂ incubator, then washed once with PBS and metabolism buffer ( metabolic inhibition buffer; 106 mM NaCl, 4.4 mM KCl, 1 mM MgCl _{2 ·} 6H ₂ O, 38 mM NaHCO ₃ , 2.5 mM CaCl ₂ , 20 mM 2-deoxyglucose, 1 mM NaCN, pH 6.6) The treatment was varied by time.

While inducing chemical ischemia as described above, while observing the degree of cell damage under a microscope from time to time, the cells at the time of the titration damage were washed twice with 1 ml of PBS, and the cells were fixed with 1 ml of 3.7% formaldehyde. . The immobilized cells were washed with 1 ml of PBS, stained with 0.1 μg / ml of DAPI (Molecular Probes) for 30 minutes, washed again three times with PBS, and killed by fluorescence microscopy (OLYMPUS 1X71, OLYMPUS). The ratio of was observed and MOCK cells that did not induce chemical ischemia were analyzed in the same manner as above. After repeating the experiment three or more times, the average ratio of dead cells 90 minutes after induction is shown in FIG. 1.

(1-3) Cell viability measurement

In order to measure the cell viability of cells induced with chemical ischemia, the following XTT assay was performed.

The transformed H9c2 cells of (1-1) were transferred to 96-well plates, incubated for 48 hours at 37 ° C., CO ₂ incubator, and chemical ischemia was induced in the same manner as in (1-2). After 90 minutes, XTT solution (Promega) was added to each well, and when color developed, the absorbance was measured at 490 nm using an ELISA plate reader (Emax, Molecular Devices). The cell viability was observed by comparing the color development of MOCK cells that did not induce chemical ischemia to 100, and the average values of three or more cycles are shown in FIG. 2.

As shown in FIGS. 1 and 2, ischemic cell death was significantly increased in FAF1 overexpressed cells during ischemia induction compared to MOCK cells, and cell survival rate was decreased due to overexpression of FAF1, indicating that FAF1 activates ischemic cell death. have.

Example 2: Confirmation of siRNA-FAF1 Inhibition of FAF1 Protein Expression

In order to confirm that siRNA against FAF1 gene inhibits the expression of FAF1 protein, the following experiment was performed.

(2-1) Construction of siRNA-FAF1

SiRNA-FAF1 corresponding to the nucleotide sequence of the human FAF1 protein and the nucleotide sequence of the rat FAF1 protein, respectively, were prepared by the following method.

First, using the siRNA Design Tool program of QIAGEN, SEQ ID NOs: 3 and 4 corresponding to bases corresponding to positions 526-544 from the first base of the start codon of the human FAF1 protein having the nucleotide sequence of SEQ ID NO: 2 The forward and reverse single-stranded siRNA-human FAF1 (hFAF1) sequences of human FAF1 having the nucleotide sequence of were synthesized, and were positioned at positions 548-564 from the first base of the start codon of the rat FAF1 protein having the nucleotide sequence of SEQ ID NO: 9. Forward and reverse single-stranded siRNA-rat FAF1 (rFAF1) sequences of rat FAF1 having the nucleotide sequences of SEQ ID NOs: 5 and 6 corresponding to the corresponding bases were designed and synthesized by QIAGEN.

40 μM of the forward and reverse single-stranded siRNA pairs synthesized as described above were added to annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4) and 2 mM magnesium acetate), respectively, and reacted at 90 ° C. for 1 minute. Then, the reaction was further performed at 37 ° C. for 1 hour to prepare double-stranded siRNA-hFAF1 and siRNA-rFAF1.

(2-2) Western Blot Analysis

In order to confirm the expression level of the FAF1 protein by double-stranded siRNA-hFAF1 and siRNA-rFAF1 prepared in (2-1), Western blot using a monoclonal anti-FAF1 antibody (Dr. Lim Suk-suk of Sookmyung Women's University) The analysis was performed as follows.

Double stranded siRNA hFAF1 and scRNA (scrambled RNA, QIAGEN) on human HeLa cells (ATCC, CCL-2) (see Zhang, L. et al, J. Biol. Chem. 279: 2053-2062, 2004) Were transformed in the same manner as in (1-1) of Example 1, and then transformed cells were incubated for 72 hours at 37 ° C. in a CO ₂ incubator. After collecting the cultured cells, using an ultrasonic powder crusher (Sonic & Materials Co., Ltd.) was subjected to ultrasonic grinding for 1 minute at Output 5, Duty cycle 30, and then centrifuged at 13,000 rpm, 4 ℃ for 10 minutes, each Cell lysate fractions (30 μg) were electrophoresed on SDS-PAGE gels, followed by Western blot analysis using monoclonal anti-FAF1 antibody and anti-β-tubulin antibody (Sigma) to express FAF1 protein. The levels were confirmed and the results are shown in FIG. 3.

In addition, the rat H9c2 cells were transformed with double-stranded siRNA-rFAF1 and scRNA in the same manner as in Example 1-1-1, and then the transformed cells were cultured for 24, 48, 72 and 96 hours. Western blot analysis was performed in the same manner as above except for the results.

As shown in Figs. 3 and 4, the expression of FAF1 decreased when both siRNA-hFAF1 was treated to HeLa cells and when siRNA-rFAF1 was treated to H9c2 cell line, indicating that siRNA-FAF1 inhibited the expression of FAF1 protein. Able to know.

Example 3 Confirmation of Inhibition of Ischemic Cell Death of siRNA-FAF1

In order to determine whether siRNA-FAF1 inhibits ischemic cell death in cells, the following experiment was performed.

(3-1) Observation of ischemic cell death

Example 1 except that HeLa cells were transformed with MOCK (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA, and double-stranded siRNA-hFAF1 prepared in Example 2 (2-1), respectively. After transforming HeLa cells in the same manner as in (1-1) and (1-2), chemical ischemia was induced, and after 90 minutes, DAPI staining was performed in the same manner as in Example 1 (1-2). The percentage of dead cells was observed, and the average value of three or more experiments is shown in FIG. 5.

In addition, except that H9c2 cells were transformed with MOCK (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA, respectively, and the double-stranded siRNA-rFAF1 prepared in Example 2 (2-1). In the same manner, the percentage of dead cells due to chemical ischemia was observed, and the average value of three or more experiments is shown in FIG. 6.

As shown in FIGS. 5 and 6, overexpression of FAF1 enhances ischemic cell death, whereas inhibition of expression of FAF1 by siRNA-FAF1 inhibits ischemic cell death, suggesting that FAF1 protein is essential in ischemic cell death.

(3-2) FACS Analysis

Example 1-1 and (1) except that the H9c2 cells were transformed with MOCK (pFlag-CMV-5a) plasmid DNA and siRNA-rFAF1 prepared in Example 2 (2-1). After the transformation in the same manner as 1-2), chemical ischemia was induced. After 90 minutes, each cell was washed once with PBS, followed by FITC binding-annexin V antibody (BD) for distinguishing apoptosis and propidium iodide (PI, sigma) for distinguishing cell necrosis. ), And analyzed using a flow cytometer (fluorescence activated cell sorter (FACS), BD) and compared the number of PI-positive cells, the results are shown in Figures 7 and 8, respectively.

As shown in FIGS. 7 and 8, siRNA-FAF1 suppresses apoptosis and cell necrosis in ischemic cell death, and thus, FAF1 plays an important role in ischemic cell death.

Example 4 Observation of DEF Formation of FAF1 in Ischemic Cell Death

FAF1 is a new member of DEF and has been reported to enhance anticancer-induced cell death by increasing the formation of DEF (see Park, MY et al, Int. J. Cancer. 115: 412-418, 2005). . Therefore, we investigated whether FAF1 enhances cell death through the regulation of DEF formation in ischemic cell death as follows.

MOCK (pFlag-CMV-5a) and pFlag- with GFP-Casease-8-DEDs (see Park, MY et al, Int. J. Cancer. 115: 412-418, 2005) in H9c2 cells . H9c2 in the same manner as in (1-1) and (1-2) of Example 1, except that the hFAF1 plasmid DNA and siRNA-rFAF1 prepared in (2-1) of Example 2 were transformed After the cells were transformed, chemical ischemia was induced. After 90 minutes the cells were washed twice with 1 ml PBS and then immobilized with 1 ml 3.7% formaldehyde. They were washed again with 1 ml of PBS, stained with PI, then washed three times with PBS, and observed the formation of intracellular DEF under confocal microscopy (Bio-rad). Shown in

As shown in FIG. 9, overexpression of FAF1 during ischemic cell death increases the aggregation of DEF formed by GFP-Casease-8-DEDs, whereas siRNA-FAF1 inhibits the increase in DEF formation, thereby ischemic cell death. It can be seen that FAF1 regulates the formation of DEF in the process.

Example 5: Confirmation of the relationship between FAF1 and caspase cell death pathways in ischemic cell death

To investigate whether FAF1 is via the caspase cell death pathway in ischemic cell death, M9 (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA in H9c2 cells, and siRNA prepared in (2-1) of Example 2 above -rFAF1 was transformed in the same manner as in (1-1) of Example 1, respectively. The transformed cells were incubated for 36 hours at 37 ° C., CO ₂ incubator and then treated or untreated with z-VAD-fmk, an inhibitor of all caspases, at a concentration of 10 μM for 12 hours. The cells were induced with chemical ischemia in the same manner as in Example 1 (1-2), and 90 minutes later, the percentage of dead cells was obtained by DAPI staining in the same manner as in Example 1 (1-2). It was observed, and the average value performed more than three times is shown in FIG.

As shown in FIG. 10, FAF1 overexpression increased cell death at normal conditions or at ischemia regardless of whether the caspase activity inhibitor was treated, whereas siRNA-FAF1 induced cell death regardless of the caspase activity inhibitor. Inhibition suggests that FAF1 is independent of the caspase cell death pathway.

Example 6: Confirmation of the relationship between FAF1 and reactive oxygen species (ROS) generation in ischemic cell death

The production of reactive oxygen species upon induction of ischemia has already been reported (see Bonne, C. et al., Gen. Pharmacol. 30: 275-280, 1998). Therefore, in order to investigate whether FAF1 regulates the production of reactive oxygen species in ischemic cell death, MOCK (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA were prepared in H9c2 cells, and Example 2 (2-1). Chemical ischemia was induced after H9c2 cells were transformed in the same manner as in (1-1) and (1-2) of Example 1, except that siRNA-rFAF1 was transformed, respectively. After 90 minutes, 5 μM of DCF-DA (2 ', 7'-dihydrodichlorofluorescindeacetate, Molecular Probes), which is an indicator of reactive oxygen species generation, was treated for 30 minutes and analyzed by a spectrophotometer (VICTOR3, Perkin Elmer). An average value of three or more times is shown in FIG. 11.

As shown in FIG. 11, overexpression of FAF1 in ischemic apoptosis induces the production of reactive oxygen species in cells, whereas siRNA-FAF1 inhibits the production of reactive oxygen species, and thus, in vivo ischemic cell death through FAF1. It can be seen that the generation of.

Example 7: Confirmation of the relationship between FAF1 and PARP-1 activation in ischemic cell death

It has already been reported that the generation of reactive oxygen species promotes the activation of PARP-1 (see van Wijk, SJ et al., Free Radic. Biol. Med. 39: 81-90, 2005), FIG. 11 As shown in, FAF1 increases the production of reactive oxygen species in cells during ischemia, and therefore, whether FAF1 regulates PARP-1 activity was examined as follows.

Example 1 except that the H9c2 cells were transformed with MOCK (pFlag-CMV-5a) and pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1 prepared in Example (2-1). Chemical ischemia was induced after H9c2 cells were transformed in the same manner as in 1-1) and (1-2). After 90 minutes the cells were disrupted to obtain each cell lysate fraction, which was then added to a histone coated plate to which PARP-1 binds. After the reaction was performed for 30 minutes by adding the substrate solution of the colorimetric quantitative analysis kit (Trevigen) and the reaction buffer to the plate, the activity of PARP-1 was measured by measuring the absorbance at 650 nm using an ELISA plate reader (Molecular Devices). Was evaluated, and the average value of three or more times is shown in FIG. 12.

As shown in FIG. 12, overexpression of FAF1 in ischemic apoptosis induces activation of PARP-1, whereas siRNA-FAF1 inhibits activation of PARP-1 in ischemia, so FAF1 increases PARP-1 activity in ischemic apoptosis. It can be seen.

Example 8: Confirmation of FAF1 and AIF Nuclear Migration in Ischemic Cell Death

Increased PARP-1 activity has been reported to release AIF protein from the mitochondria and transfer it to the nucleus (see van Wijk, SJ et al., Free Radic. Biol. Med. 39: 81-90, 2005). As shown in FIG. 12, since FAF1 is involved in increasing PARP-1 activity, whether FAF1 is involved in nuclear migration of AIF was investigated as follows using fractionation analysis.

To determine the effect of ischemic cell death on the positional shift of AIF acting in PARP-1 subregion, H9c2 cells were transformed with MOCK (pFlag-CMV-5a), pFlag-hFAF1 plasmid DNA, and siRNA-rFAF1, respectively. Except that except for transforming H9c2 cells in the same manner as in (1-1) and (1-2) of Example 1 to induce chemical ischemia. After 90 minutes, the cells were washed twice with 5 ml of PBS, then fractionated cytolysis buffer (250 mM sucrose, 20 mM HEPES, pH 7.5), 10 mM KCl, 1.5 mM MgCl ₂ , 1 mM EDTA, 1 mM EGTA , 1 mM DTT and 0.1 mM PMSF) were added and left on ice for 15 minutes, and the precipitated cells were disrupted using a 1 ml syringe. After centrifugation at 1,000 g for 15 minutes, the precipitate was taken to obtain a nuclear fraction, and the supernatant was further centrifuged at 15,000 g for 20 minutes, and the precipitate was taken to obtain a mitochondrial fraction. These nuclei and mitochondrial lysates (20 μg) were electrophoresed on 8% SDS-PAGE gels and then subjected to Western blot analysis using monoclonal anti-AIF antibody (E1, Santa Cruz). At this time, in order to confirm the correct fraction of the nucleus and mitochondria, polyclonal anti-PARP-1 antibody (H-300, Santa Cruz) and polyclonal anti-SOD, which are antibodies of PARP-1 and SOD-2, respectively, marker proteins -2 (HL-222, Santa Cruz) was used, and the results are shown in FIG.

As shown in FIG. 13, AIF in the cytoplasm shifts to the nucleus during ischemic induction, and nuclear migration of AIF is inhibited by siRNA-FAF1, so that FAF1 affects AIF nuclear migration in ischemic cell death. .

Example 9 Confirming Ischemic Cell Death Protective Effect of Dominant-Negative FAF1

To identify the influence of dominant negative (DN) FAF1 on ischemic cell death, expression vectors MOCK (pFlag-CMV-5a), pFlag-hFAF1 and pFlag-hFAF1 [366-650] (FAF1- ΔFID.DEDID, DN, see Ryu, SW et al., J. Biol. Chem. 278: 24003-24010, 2003) Example 1-1 above except that the plasmid DNA was transformed. H9c2 cells were transformed in the same manner as in () and (1-2), chemical ischemia was induced, and then the percentage of dead cells was observed by DAPI staining. .

As shown in FIG. 14, ischemic cell death was inhibited when the function of FAF1 was inhibited by dominant-negative FAF1. Thus, it can be seen that FAF1 is an essential protein in the ischemic cell death process.

From these results, FAF1 acts on caspase-independent cell death pathway, and ischemic cell death FAF1 plays an important role in ischemic cell death process through the generation of reactive oxygen species, PARP-1 activation and nuclear migration pathway of AIF. Able to know.

As mentioned above, the human FAF1 protein inhibitors of the present invention, such as siRNA and dominant-negative FAF1 protein, effectively inhibit the expression or function of FAF1 protein, generate ischemic cell death caspase-independent cell death pathway, and generation of reactive oxygen species. Because it effectively inhibits ischemic cell death by acting on PARP-1 activation and nuclear migration pathway of AIF, the pharmaceutical composition according to the present invention containing the inhibitor as an active ingredient is cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, myocardium It can be effectively used for the treatment or prevention of ischemic diseases such as hypertrophy, retinal ischemia, ischemic colitis and ischemic acute renal failure.

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Claims (13)

A double stranded siRNA consisting of a forward single stranded siRNA having a nucleotide sequence of SEQ ID NO: 3 and a reverse single stranded siRNA of SEQ ID NO: 4, Double-stranded siRNA consisting of a forward single-stranded siRNA having the nucleotide sequence of SEQ ID NO: 5 and a reverse single-stranded siRNA of SEQ ID NO: 6, and Selected from the group consisting of proteins having the amino acid sequence of SEQ ID NO: 7 A composition for the treatment or prevention of ischemic diseases, which contains a substance that inhibits the expression or function of human FAF1 (Fas-associated factor 1). delete delete delete delete delete The method of claim 1, The ischemic disease is selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis and ischemic acute renal failure mediated by ischemic cell death. A method for targeting FAF1 mRNA using a double-stranded siRNA consisting of a forward single-stranded siRNA having the nucleotide sequence of SEQ ID NO: 3 and a reverse single-stranded siRNA of SEQ ID NO: 4, A method of targeting FAF1 mRNA using a double stranded siRNA consisting of a forward single stranded siRNA having the nucleotide sequence of SEQ ID NO: 5 and a reverse single stranded siRNA of SEQ ID NO: 6, and Selected from the group consisting of a method of binding a protein having an amino acid sequence of SEQ ID NO: 7 with FAF1 to inhibit physiological activity mediated by FAF1, A method of inhibiting ischemic cell death by inhibiting the expression or function of FAF1 protein in mammals other than humans in need of suppression of ischemic cell death. delete delete delete delete delete

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