KR101168054B1 - Pharmaceutical Composition for Preventing or Treating Autophagy-associated Disease and Method of Screening Thereof - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising: (a) a pharmaceutically effective amount of a small molecule named autophagonizer or a pharmaceutically acceptable salt thereof; And (b) relates to a pharmaceutical composition for the prevention or treatment of autophagy-related diseases comprising a pharmaceutically acceptable carrier.
The pharmaceutical composition comprising the autophagogenizer of the present invention effectively induces autophagy of cells, thus exhibiting great efficacy in preventing or treating autophagy-related diseases. In addition, the pharmaceutical composition comprising the autophagogenizer of the present invention induces cell death by apoptosis-independent mechanism, and thus can be effectively used even when the apoptosis mechanism is impaired.

Description

Pharmaceutical Composition for Preventing or Treating Autophagy-associated Disease and Method of Screening Thereof}

The present invention relates to a pharmaceutical composition for preventing or treating autophagy-related diseases. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating autophage-related diseases, including a small molecule named autophagogenizer.

Autophages play an important role in regulating cellular functions such as starvation, protection from infectious bacteria and regulation of neurodecay. Autophagy is an evolutionarily conserved process that occurs in all eukaryotes, from yeast to mammals (Klionsky et al. (2000)). Early in autophagy, cytoplasm and intracellular organs degenerate into a double-membrane-bound structure called an autophagosome. The autopago island is then fused with the lysosome to form an autolysosome, and the degraded material is degraded by lysosomal hydrolase and reused. However, the molecular mechanisms inherent in this process are not yet known (Tsukada and Ohsumi (1993); Thumm et al. (1994)).

The autophagy pathway comprises two unique ubiquitin-like conjugation systems: Atg8PE (phosphatidylethanolamine) and Atg12Atg5. This pathway is involved in the formation of autophago islands with the class III phosphatidylinositol 3-kinase (PI3K) complex (Kihara et al. (2001)), an East Vps34 homologue (Ohsumi (2001)). Beclin 1 (ortholog of yeast Atg6) in the complex is a cancer suppressor gene product (Liang et al. (1999); Qu et al. (2003); Yue et al. (2003)). In many cell types, stimulation of the class I PI3K / AKT signaling pathway by growth factors and cytokines has the effect of inhibiting autophagy (Petiotet al. (2000); Melendez et al. (2003); Arico et al. (2001); Kanazawa et al. (2004). The inhibitory effect is probably due to class I PI3K / AKT-dependent activation of the target of rapamycin (TOR) kinase that downregulates autophagy (Blommaart et al. (1995); Noda and Ohsumi (1998); Abeliovich et al. (2000); Abeliovic et al. (2003). Cells treated with the PI 3-kinase inhibitors wortmannin and 3-methyladenine (3-MA) do not generate autophagosome precursors, which are active during the early stages of autophagy vacuogenesis. This is important (Mizushima et al. (2001). In fact, 3-MA has long been used as an autophagy inhibitor (Seglen and Gordon (1982)).

It is recently known that autophagy can cause cell death under certain conditions. Cell death is generally associated with apoptosis, but may also occur in other ways, such as autophagy and non-lysosomal vesicular cell death. However, the mechanism inherent in autophagy cell death is little known. Numerous studies report that autophagy or autophagy cell death is activated in response to various chemotherapy in cancer cells derived from breast, colon, prostate and brain.

In particular, many cancer cells are resistant to malnutrition because of their ability to activate autophagy (Hwang and Lee (2007)). Some cells carry mutations in the ATG gene, such as the gene encoding the cancer suppressor Becklin 1. Autophagy promotion results in inhibition of cancer cell growth and cell death.

Thus, new and potential autophagy-inducing substances can greatly contribute to studying the role of autophagy in tumor formation and can form the basis of new cancer therapies.

Various physicochemical therapies have been reported to induce autophagy in cancer cells: exposure to oncogene Ras (Chi et al. (1999)), irradiation (Paglin et al. (2001), arsenic trioxide (Kanzawa) et al. (2005)), ceramides (Daido et al. (2004)), resveratrol (Opipari et al. (2004)), temozolomide (Kanzawa et al. (2004)) Dopamine (Gomez-Santos et al. (2003)), endostatin (Tal-Or et al. (2003)), soybean B-group triterpenoid saponins (Ellington et al. (2005)) and histone deacetylases (HDAC) inhibitors (Shao et al. (2004); Oh et al. (2008)).

Rapamycin, an inhibitor of the mammalian rapamycin target (mTOR), induces autophagy and inhibits the proliferation of malignant glioma cells (Takeuchi et al. (2005)). The fact that autophagy plays a key role in controlling cancer progression is that 40-75% of beclin 1 has been deleted as monoallele in sporadic breast, ovarian and prostate cancers, and the beclin 1 is in mice. It is supported by recent research showing that it functions effectively as a monodeficient cancer inhibitor (Qu et al. (2003); Yue et al. (2003)). The mechanism by which autophages provide a tumor-forming barrier is still theoretical, and the signaling pathways involved from autophagy to cell death are not yet fully known.

In atherosclerosis, treatment with rapamycin-coated stents significantly reduced the number of adherent macrophages but did not remove muscle cells (Verheye et al. (2007)). Macrophages play a central role in plaque removal, and the fact that these macrophages can be selectively purified by autophagy cell death suggests a new treatment for atherosclerotic plaques. Autophagy can also increase aging-related diseases. Age-related macular degeneration (AMD) can occur as a result of exosome activation in autophagy and aged retinal pigment epithelial cells (Wang et al. (2009)). AMD is the leading cause of visual loss in middle-aged drusen patients, and extracellular amorphous accumulation of substances in the retinal macular area is an early signal of AMD. Druzen refers to a white or yellow extracellular substance that accumulates in the eye brunch membrane. Drusen in the AMD donor eye contains the autophagy markers Atg5, Atg12-Atg5 conjugates and exosomes, supporting the fact that the increase in internal proteins secreted by autophagy and exosomes contribute to drusen formation. . The regulation of autophagy is an important factor in controlling intracellular homeostasis and thus forms the basis for potential treatment of serious diseases such as cancer, atherosclerosis and AMD.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a novel pharmaceutical composition that can effectively prevent or treat autophagy-related diseases. As a result, a small molecule named autophagonizer is apoptotic-independent mechanism. The present invention has been completed by finding that it effectively induces autophagy.

Accordingly, an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of autophagy-related diseases.

Another object of the present invention to provide a method for screening or preventing autophagy-related diseases.

Still other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention is (a) a pharmaceutically effective amount of a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; And (b) provides a pharmaceutical composition for the prevention or treatment of autophagy-related diseases comprising a pharmaceutically acceptable carrier:

Formula 1

IUPAC name of the compound represented by Formula 1 of the present invention is 2- (3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo [4,5] thieno [2,3- d] pyrimidin-2-ylsulfanylmethyl) -oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide, and the inventors named it autophagonizer. The autophagogenizer effectively induces autophagy of cells. Autophagy is an action to protect cells from environmental imbalances and pathogens.

In particular, the autophagogenizer induces autophagy of hyperproliferative cells in a mechanism independent of apoptosis. Many cancer cells survive through mutations that inactivate apoptosis mechanisms or activate anti-apoptotic pathways, resulting in resistance to traditional chemotherapeutic agents that utilize apoptosis mechanisms. Since the autophagogenizer acts as an apoptosis-independent mechanism, it can be effectively used for the prevention and treatment of apoptosis-resistant hyperproliferative diseases.

In addition, the autophagogenizer of the present invention is characterized by inducing autophagy of hyperproliferative cells as a mechanism for upregulating BHMT (betaine-homocysteine methyltransferase) expression. BHMT is an autophagosomal membrane enzyme required for glutathione (GSH) synthesis and is known as a target of 3-MA (Furuya et al. (2001)). 3-MA prevents loss of BHMT activity, resulting in a decrease in intracellular GSH / GSSG ratio. BHMT is an enzyme rich in liver and kidney that catalyzes the transfer of methyl groups from betaine to homocysteine to produce dimethylglycine and methionine (Lee et al. (1992); Garrow (1996)). Cytosol BHMT is ingested during autophagy degeneration and converted to p32, a 32-kDa fragment, which is closely related to the fusion of autophagosomes and lysosomes. Thus, an increase in BHMT levels in the cell, together with the formation of autophagy vacuoles, is a new sign of autophagy.

In one embodiment, the effective amount of autofaginizer is included at a concentration of 1 to 1000 in the total pharmaceutical composition.

The autophagogenizers of the present invention may be included in the compositions of the present invention in the form of the compounds of formula (I) as well as pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound.

The pharmaceutical composition of the present invention is particularly effective for the treatment and prevention of autophagy related diseases. Autophagy-related diseases are diseases that can be treated by inducing autophagy. Specifically, autophagy inhibits cell growth and differentiation, reduces mutagenesis, or removes cell organelles such as mitochondria damaged by reactive oxides. All diseases that can be treated by doing so. Examples of autophagy-related diseases include cancer, atherosclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, and ocular pharyngeal dystrophy. ), Prion disease, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral pallidoluysian atrophy, frontal lobe dementia, progressive supranuclear palsy, x-linked spinobulbar muscular atrophy and neuronal intranuclear hyaline inclusion disease.

Specific examples of the cancer among autophagy-related diseases include pituitary adenoma, glioma, brain tumor, glandular cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, and gallbladder cancer. , Penile cancer, ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytoma, leukemia, childhood cancer, skin cancer, ovarian cancer or cervical cancer.

As used herein, the term “treating”, unless stated otherwise, reverses, alleviates, or progresses the disease or condition to which the term applies, or one or more symptoms of the disease or condition. It means to inhibit or prevent. As used herein, the term "treatment" refers to the act of treating when "treating" is defined as above.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be oral or parenteral, and parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration and the like. It is also possible to administer the compound in a local rather than systemic manner, for example by direct injection into hyperproliferative cells, often in immersion or sustained release formulations.

The formulation of the composition may vary depending on the method of use, but may be used as a warning agent (Plasters), granules (Granule), powders (Sowrups), syrups (Syrups), solutions (FluidextractsI), emulsions (Emulsions) , Suspensions, infusions, tablets, injections, capsules and pills. Proper formulation is dependent upon the route of administration chosen. Any of the known techniques, carriers and excipients can be used suitably and as understood in the art, for example in Remingston's Pharmaceutical Sciences described above.

In addition, the dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the age, sex, condition, absorption of the active ingredient in the body, inactivation rate and the drug used in combination, based on the compound of Formula 1 When administered, it may be administered at 0.001 μg / kg body weight to 500 mg / kg body weight.

According to another aspect of the present invention, the present invention comprises the steps of (a) treating a test substance to a cell comprising a beta-homocysteine methyltransferase (BHMT) nucleotide sequence; And (b) analyzing the expression of the BHMT, and when the test substance increases the expression of the BHMT, it provides a screening method for preventing or treating autophage-related diseases, which is determined to be an autophagy-related disease prevention or treatment.

Autophagy is regulated by several signal pathways, and the inventors have found that autophagy induced by the treatment of the autophagogenizer is regulated through a novel signal pathway, which is characterized by significant upregulation of BHMT. I found out that I was losing. In other words, not only the maturation of autophagy vacuoles or the conversion of LC3-I to autophagosome-binding LC3-II, but also significant upregulation of BHMT is a sign of autophagy.

In one embodiment of the invention, the BHMT gene is a nucleotide sequence of the gene registration number (GenBank) BC012616.

Analyzing the expression of BHMT can be carried out in a variety of ways, in particular can be carried out using methods known in the art. For example, RNA is isolated from a test cell treated with a test substance and a control cell not treated with a test substance to a cell including a BHMT nucleotide sequence, from which a cDNA is synthesized and labeled with different fluorescent substances. Thereafter, the labeled cDNA may be hybridized with the genes of the BHMT protein, and the expression level of the BHMT may be analyzed by comparing the degree of BHMT gene expression between the test group and the control group, for example, by using RT-PCR. Expression can be analyzed.

The features and advantages of the present invention are summarized as follows:

(Iii) the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a small molecule or pharmaceutically acceptable salt thereof; And (b) provides a pharmaceutical composition for the prevention or treatment of autophagy-related diseases comprising a pharmaceutically acceptable carrier.

(Ii) Since the composition of the present invention effectively induces autophagy of cells, it exhibits great efficacy in preventing or treating autophagy-related diseases. In addition, the composition of the present invention induces the death of hyperproliferative cells in a mechanism independent of apoptosis, and thus can be effectively used even when the apoptosis mechanism is impaired.

Figure 1. Autofaginizer Identification and Screening Assay
(A) The structure of the autopaginizer.
(B) Dot pattern of increased GFP-LC3 expression in terms of size and density in cells treated with (a) untreated control and (b) 10 μM autopagogenizer in cell-based screening assay. Accumulation of Lysotrack Red in MCF-7 cells cultured for 24 hours with or without 10 μM autopagogener ((c): control, (d): autopagoneizer) and MDC-labeled vacuole ((e): A control group (f): autophagogenizer) was observed under a fluorescence microscope.
(C, D) High-Dose Screening Analysis of Autopagonaser Treated Cells. Total fluorescence intensity (C) per well of each sample and total fluorescence intensity (D) per cell of each sample were measured. R stands for rapamycin and A stands for autopaginizer. Values are expressed as mean ± SE. Scale bars indicate 20 μm.
Figure 2. Cytotoxicity effects of auto digging homogenizer; And Ultrastructural Properties and LC3 Conversion Western Autophagy Activity Detection Using Blots
(A) Amount-dependent efficacy of autophagogenizers. Cells were treated with 0, 1, 2, 5 or 10 μM autopaginizer for 2 days and cell viability was estimated by MTT assay.
(B) reduced survival after autopagogenizer treatment. HT1080 cells were treated with 0, 1, 2, 5 or 10 [mu] M autophagogenizer for various times and cell viability was estimated by MTT assay. Values from three independent experiments are expressed as mean ± SE.
(C, D) The ultrafine structure of cells cultured for 1 day in the absence of (C) and (D) of μM autopagogenizer is shown by TEM. Arrows point to representative autophagy vacuoles containing nearly complete organs and digested residual material. Scale bar: 1 μm.
Figure 3. HT1080 cells LC3 -I of LC3 - II observed the conversion of the
In order to monitor the conversion of LC3-I to LC3-II in (E, F) autofagonizer-treated HT1080 cells, cells were treated with 10 μM autopagogenizer or camptothecin, or 20 μg / mL rapamycin for 24 hours. Treated. Immunoblots using anti-LC3 antibodies were performed in equal portions of the protein extracts. Tubulin was used as an internal control. (E) Significant increases in LC3-II were observed both with autophagogenizer and rapamycin. (F) Quantitative analysis of increased LC3 conversion. NT: untreated, A: autopagogenizer, C: camptothecin, and R: rapamycin.

Figure 4. Cell death is automatic digging in HT1080 cells induced by a homogenizer is independent of apoptosis.
(A) Hoechst 33342 staining of apoptotic nuclei. (B) DNA fragment analysis using agarose gel electrophoresis. (C) Quantification of apoptosis. Cells were treated with 10 μM autopagogenizer or camptothecin for 24 hours, and apoptosis was analyzed by FACS and Annexin V-FITC staining. (D) Influence of carspace inhibitors. Prior to treatment with cells for 10 hours with 10 μM autopagogenizer or camptothecin, the cells were pre-treated with DMSO or carspace inhibitor Z-VAD-FMK (50 μM) for 1 hour. Control cells were treated only with DMSO. The percentage of cells that did not survive was estimated by trypan blue staining. Values from three independent experiments are expressed as mean ± SE.
Figure 5. Auto Auto digging phage induced by the homogenizer is associated with an unknown mechanism.
Prior to 24 hours treatment of cells with 10 μM autophagogenizer, the cells were pretreated for 1 hour with DMSO or autophagy inhibitors 3-MA (10 mM), wortmannin (100 nM), PD98059 (10 μM). The percentage of cells that did not survive was estimated by trypan blue staining. Values from three independent experiments are expressed as mean ± SE.
Figure 6. Dual Signal Rank Cluster
Each double signal class cluster is shown under two experimental conditions. Scattered plots and signal correlations (ρ) between the two experimental conditions (autopagonizer vs. rapamycin) show low correlation.

Figure 7. RT - PCR proof of autophagogenizer -regulatory genes
In autophagogenizer-treated cells, BHMT and Becklin 1 RNA levels increased in a time dependent manner and BIRC levels decreased in a time dependent manner. (B) Quantitative analysis of RNA levels of BHMT, Becklin 1 and BIRC.
Figure 8. Fargo Auto Ionizer - Effect of azithromycin bar Philo A1 to induce auto-grip.
(A) AVO quantitation using FACS scan and acridine orange. Twenty four hours after treatment with 10 [mu] M autopagogenizer, cells were exposed to parasitic staining acridine orange. For the combination treatment, cells were treated with 200 nM batillomycin A1 1 hour prior to treatment with 10 μM autopagogenizer. Control cells were treated with PBS only. FL1-H and FL3-H indicate green and red intensities, respectively. (B) Acridine orange staining after autopagogenizer and bacillomycin A1 treatment on HT1080 cells. Scale bars indicate 20 μm.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Experiment Method and Materials

Cell Culture and Screening Assays

AGS (human gastric epithelial tissue), HCT116 (human colorectal carcinoma), A549 (human lung carcinoma), MCF7 (human epithelial tissue-like mammary carcinoma), HeLa (human epithelial carcinoma) and COS7 (human apes virus 40-transforming African green) Monkey kidney) cells were cultured and preserved in DMEMbe's modified Eagle's medium containing 10% fetal bovine serum (FBS). HT1080 (human fibrosarcoma) cells were incubated and preserved in MEM (Minimal Essential Medium) medium containing 10% FBS. According to the Invitrogen protocol, COS7 cells were transformed with pEGFP-LC3 with Lipofectamine 2000 to prepare stable COS7 cell lines expressing EGFP-LC3. Stable clones were selected in complete medium containing G418 750 / mL. pH 7.4, 5% CO ₂ Cells were incubated at ambient and 37 ° C. COS7 cells stably expressing EGFP-LC3 were treated with the compound for 24 hours, and then observed under a fluorescence microscope.

Compounds and Plasmids

The 2000 compounds used for the screening of the present invention were obtained from ChemDiv, Chemgenex and Korea Chemical Bank, and included structure-based synthetic libraries and natural product libraries. The compounds were stored in 10 mM DMSO stock at -80 ° C. Plasmids and anti-LC3 antibodies encoding EGFP-LC3 were provided by Tamochus Yoshimori (National Institute for Basic Biology, Okazaki, Japan).

reagent

DMEM, MEM and FBS were obtained from Gibco Laboratories (Grand Island, NY). Hoechst 33258, LysoTracker and FITC-conjugated Annexin V were obtained from Molecular Probe (Eugene, OR). Carspace family inhibitor Z-VAD-FMK was obtained from Biovision (USA) and all other compounds were obtained from Sigma Chemical (St. Louis, MO).

Visualization and analysis of intracellular vacuoles

To detect autophagy vacuoles, the cells were treated with fluorescent dyes monodansylcadaverine (MDC) 50, known to accumulate in autophagy organs (Biederbick et al. (1995)). After incubation the cells were washed four times with PBS and immediately analyzed by fluorescence microscopy. Cells were labeled with 50 nM Lysotrack Red for 20 minutes to analyze the acidic compartments of the cells, and cells were observed using fluorescence microscopy after one wash and media exchange. Lysotrack Red is a fluorescent dye that accumulates specifically in acidic organs and primarily labels functional lysosomes (Stefanis et al. (2001)).

High-Content Screening Analysis

Quantitative fluorescence analysis of stained autophagy vacuoles was performed using CellomicsArrayScanVTI (Thermo Scientific). The target activation protocol of Thermo Scientific High-Content Informatics (HCi ™) was used to measure the fluorescence intensity of nuclei and cytosolic fibrils. Cells were placed in 96-well plates and treated with an autopaginator for 24 hours. Nuclei and autophagy vacuoles were then stained using Hoechst 33342 (1: 1000) and Lysotrack Red, respectively. Each cell was identified using Hoechst 33342 staining of the HCi ™ Target Activation Protocol, and the cell area surrounding the nuclei was measured with LysoTrack Red according to the manufacturer's protocol.

Cell Survival and Death Measurement

Cell growth was measured using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) colorimetric assay. Various cancer cells were fixed in 96-well plates at a density of 5 × 10 ³ cells / well and incubated for 24 hours for stabilization. The cells were treated with various concentrations of autophagogenizer at various time points. At the end of the assay 2 mg / ml MTT was added to each well and incubated for 4 hours. MTT-formazan of each well was dissolved in 50 μl DMSO and absorbance was measured at 540 nm using a microplate reader (Bio-Tek Instrument Inc., Winooski, VT).

Electronic transmission microscope Transmission Electron Microscopy )analysis

Trypsin treatment yielded cells, washed twice with PBS and fixed cells with 2% paraformaldehyde / 2% glutaraldehyde in pH 7.4 and 0.1 M phosphate buffer. After fixation, cells were washed with sodium cacodylate and scraped from plates to pellet by centrifugation. Cell pellets were pre-fixed with 1% (v / v) osmic acid in sodium cacodylate and stained with 1% uranyl acetate. After dehydration, pellets were inserted into Durcopan (Fluka, Sigma). Ultra thin sections were prepared using LKB 8800 Ultratom III and observed using a Philips EM 400T electron microscope.

Hocist  By dye dyeing Apoptosis  observe

In order to observe chromatin condensation or nuclear fission, the cells were stained for 30 minutes at 37 ° C. with a 50 μg Hochest 33342 / ml culture medium, washed once and then resuspended in PBS and observed by fluorescence microscopy.

DNA  By division Apoptosis  Detection

DNA cleavage detection was performed using agarose gel electrophoresis. Cells were collected by centrifugation and washed with PBS. The washed cells were lysed with a 10 mM Tris-HCl solution at pH 8.0 containing 10 mM EDTA, 0.5% (w / v) sodium dodecyl sulfate (SDS) and 0.1% (w / v) RNase A and 50 ° C. The reaction was carried out for 1 hour at. The lysate was further reacted with 1 mg / ml proteinase K for 1 hour at 50 ° C. and 50 V in 40 mM tris acetate and 1% (w / v) agarose gel at pH 7.5 containing 1 mM EDTA. Electrophoresis was performed for 1 hour under conditions. After electrophoresis, DNA was visualized using ethidium bromide.

Annexin ( Annexin ) Using V Apoptosis  Detection

Trypsin treatment yielded cells (5 × 10 ⁵ ), which were then washed twice with PBS. The washed cells were resuspended in 200 μl binding buffer (PBS with 1 mM calcium chromide). FITC-conjugated Annexin V was added at a concentration of 0.5 μg / mL according to the manufacturer's instructions (Invitrogen, Camarillo, Calif.). After 20 minutes of reaction at room temperature, 400 μl of binding buffer was added and samples were analyzed immediately using a FAC scan (Becton Dickinson, San Jose, Calif.). Data was analyzed using Cell Quest software (Becton Dickinson).

Carspace  Inhibition assay

HT1080 cells were mounted in 6-well plates at a ratio of 2 × 10 ⁵ cells / well and reacted overnight at 37 ° C. 50 μM of Z-VAD-FMK was treated one hour prior to treatment with 10 μM autophagogenizer or 10 μM camptothecin. After 24 hours, cell survival was measured by trypan blue staining.

Western Blot  Analysis method

 Soluble protein was obtained from treated HT1080 cells dissolved in 50 mM Tris HCl buffer containing 10% glycerol, 2% SDS, 10 mM dithiothreitol and 0.005% bromo phenol blue. The same amount of protein measured by Bio-Rad protein assay was separated by 12.5% SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, Mass.). The blot was then blocked and antibody labeled overnight at 4 ° C. using anti-LC3 antibody. Labeled antibodies were visualized using enhanced chemiluminescence (ECL) kit (Amersham Life Science, Inc., Buckinghamshire, UK) according to manufacturer's instructions. Tubing was used as a loading control and was detected using anti-tubulin antibody (Upstate Biotechnology). Quantitative analysis of relative ratios was performed using ImageJ 1.40 g (NIH, USA).

Acridine  Orange staining AVOs  Quantitative and Flow Cytometry Flow Cytometry )

In acridine orange-stained cells, the cytoplasm and nucleus fluoresce bright green and dark red, respectively, while the acidic fraction emits bright red fluorescence (Garrow (1996)). Since the red fluorescence intensity is proportional to the acidity, the volume of the acidic fraction portion can be titrated (Paglin et al. (2001)). Cells were stained with acridine orange for 17 minutes, removed from the plate using trypsin-EDTA and collected in phenol red-free growth medium. Green (510-530 nm) and red (650 nm) fluorescence emitted with excitation-light of blue (488 nm) were measured by FACSCalibur (Becton Dickinson) using CellQuest software.

cDNA Microarray  And data analysis

Human 8K twin chips (Digital Genomics, Inc., Seoul, Korea) were used for transcription kinetics analysis. Test group RNA was labeled with Cy5-dUTP using reverse transcription, and control RNA was labeled with Cy3-dUTP. The labels were mounted on cDNA chips and allowed to react for 16 hours in a 65 ° C. dark water bath. The chip is cleaned, then scanned using a 428 scanner (Affymetrix, Santa Clara, CA, USA) to obtain raw data and Gensite data analysis software version 3.5 (BioDiscovery, Los Angeles, CA) Was analyzed statistically). The correlation coefficient ρ between two signals was calculated using the following equation:

Where x _k is a log ₁₀ representation of the ratio for the k ^th gene in the x signal and y _k is a log ₁₀ representation of the ratio for the k ^th gene in the y signal (Qu et al. (2003)).

RT - PCR  Analysis method

Total RNA was isolated using Trizol Reagent (Invitrogen) according to manufacturer's instructions and gene up- and down-regulation was confirmed by RT-PCR assay using the following primer pairs: 5'-CCCCATAACCCTTCTGTCA-3 for BHMT 'And 5'-ACTTTTCACAGGGTGGGTGCTTAG-3';5'-CAAGATCCTGGACCGTGTCA-3'and5'-TCGCACTTTGTGTGGACATCA-3' for Beclin 1; And 5'-CTTACGAAGAACTACGGCTGGACT-3 'and 5'-GCCATTTCTCTAAACACCCTCCAC-3' for BIRC1. Quantitative analysis of relative proportions was performed using Image J 1.40 g (NIH, Bethesda, MD).

Statistical analysis

The experimental results were expressed using the mean ± standard deviation (SD). Student's t-test was used to determine statistical significance between the control and experimental groups. p <0.05 was considered statistically significant.

Experiment result

Auto Pagonaizer New Autophagy  It is an inducer.

To identify novel autophagy inducers, a cell-root screening system using EGFP-LC3 and high-content screening were employed. LC3 is the third light chain of microtubule related protein 1 and is the key to autophagy. Because LC3-I is synthesized in the cytosolic liquid phase and converted to cell membrane-bound LC3-II during autophagy vacuogenesis, we use a COS7 cell to express a stable cell line expressing EGFP-LC3 for autophagy inducer screening. Constructed. The screening method revealed a novel autophagy inducer autopagogenizer as the most potent material in the 2,000 synthetic compound libraries (FIG. 1A). Other small molecules that were structurally similar to Autophagogenizer were also tested, but did not show strong autophagy activity. As a result of treating Cos-7 cells stably expressing EGFP-LC3 with an autophagogenizer, it was confirmed that EGFP fluorescence was increased in cytoplasm compared with untreated cells (FIG. 1B (a) and (b)). Autophagy vacuoles of MCF-7 were treated with LysoTracker Red (FIG. 1B (c), (d)) and monodansycadaverine (MDC) after treatment with autophagogenizer for 24 hours. 1B (e) and (f)) were stained, and as a result, the increase in fluorescence intensity was observed in the autophagogenizer treated cells compared to the control.

The mean fluorescence intensity of each cell and sample was measured using the Cellomics Target Activation protocol for in-depth quantitative analysis. Autophagogenizer-induced lysotrack fluorescence was 2.54 times greater per sample than control staining and about 1.5 times greater per sample than fluorescence induced by rapamycin (FIG. 1C). The mean induction rate per cell in the autophagogener treated cells was also 1.24 fold and 3.04 fold greater than in the control and rapamycin-treated cells, respectively (FIG. 1D). The results collectively indicate that the autophagogenizer is an effective inducer of autophagy.

The Auto Pagonaizer Autophagy  Decreased cell growth and viability.

Autophagogenizer-induced autophages have an effective concentration of 5 and influenced the growth of various cancer cells. MTT analysis of the cells treated with Autopagonaiser for 2 days, it was confirmed that the Autopagogonaser reduced the viability of the cells in a sheep-dependent manner (Fig. 2A).

For HT1080 fibrosarcoma cells cell viability decreased in a sheep-dependent manner over various time frames (FIG. 2B). LC3 conversion was observed using electron transmission microscopy (TEM) to demonstrate that reduced cell growth and reduced viability were the result of autophagy and not of apoptosis. After 24 hours of treatment, the autophagy vacuole was closely observed using an electron transmission microscope (TEM).

Autophagogenizer-treated cells had increased vacuole counts (FIG. 2D) compared to untreated cells (FIG. 2C). LC3 conversion during autophagical vacuogenesis was observed by immunoblot method using anti-LC3 antibody. This transformation was observed in autophagogenizer- and rapamycin-treated cells, while not in cells treated with apoptosis inducer Camptothecin or control cells (FIGS. 3E and 3F). These results indicate that autophagogener influences cell viability by inducing autophagy cell death in a pattern similar to the LC3 transformation pattern of rapamycin but not camptothecin.

Autopagoneizer Induced cell death Apoptosis and   Irrelevant

Apoptosis analysis was performed to confirm that autophagogenizer induced autophagy cell death. First, the cells were treated with autopagogenizer and camptothecin, and the nuclei were stained with Hoechst 33342. Autopagogenizer treatment resulted in fewer apoptotic cells than treatment with camptothecin (FIG. 4A).

Secondly, DNA fragment analysis showed unfragmented DNA in autophagogenerized cells and fragmented DNA fragments in camptothecin-treated cells (FIG. 4B). Third, fluorescence activated cell sorter (FACS) analysis for apoptosis quantification showed that 79.5% of the autophagogener-treated cells remained at M1, while only 50.3% of camptothecin-treated cells remained at M1. It was confirmed that there was (Fig. 4C). Finally, the number of apoptosis cells when cells were co-treated with caspase-3 and the apoptosis interferer Z-VAD-FMK was lower than in the case of camptothecin-treated cultures, but compared to autophagogener-treated cultures. There was no change (FIG. 4D). The results indicate that the autophagogenizer induces autophagy cell death in a mechanism independent of apoptosis.

The Auto Pagonaizer New Autophagy  It is related to the signal path.

Autophages are regulated by several signal pathways, and 3-MA, wortmannin and PD98059 are known to be inhibitors of these signal pathways. In order to determine which signal pathway the autophagogener acts on, cells were treated together with the autopagogenizer and the inhibitor. Cells were pretreated for 1 hour prior to autophagogenizer treatment and the number of surviving cells was counted. When cells were treated with inhibitors only, the number of dead cells decreased significantly. However, when cells were treated with both autophagogenizer and inhibitor, the number of dead cells increased due to the effect of autophagy apoptosis (FIG. 5). Since no significant changes were observed when cells were treated with inhibitor alone, the results indicate that the autophagogenizer is an autophagy inducer involved in the novel signal pathway.

In order to elucidate the mechanisms inherent in autophagogenizer-induced autophagy, cDNA microarray analysis was performed. Microarray analysis of autophagogener treated cells showed different results from rapamycin-treated cells, indicating that autophagogener induced autophagy by the rapamycin-independent pathway (FIG. 6). Autophagy- and apoptosis-related genes were upregulated and downregulated, respectively, by autophagogenizer treatment (Table 1). BHMT was most significantly upregulated, followed by autophagy-related genes, Becklin 1 and Interleukin 18. Relevant genes such as death-related protein 3 (DAP3) and Caspace 9 were downregulated, consistent with previous results. The results were confirmed by performing RT-PCR on up and down regulated genes. In autophagogenizer-treated cells, the levels of BHMT and Beclin 1 were increased while BIRC1 expression levels were decreased (FIG. 7). RT-PCR results were consistent with microarray results. The results are summarized in Table 1 below.

Batillomycin  A1 of the present invention Autopagoneizer -Judo In autophagy  Impact

Vapilomycin A1 is a known regulator of autophagy and interferes with the maturation of autophagy vacuoles. When treated with the bacillomycin A1 and autophagogenizer, the number of autophagy vacuoles was reduced (FIG. 8A). Acidic vesicular organelles (AVO) were stained with acridine orange and quantified by FACS analysis. Fluorescence intensity of AVO was reduced when the cells were treated with bacillomycin A1 prior to treatment with autophagogenizer (FIG. 8B). This result is consistent with the analysis of FACS. In conclusion, autophagogenizer treatment induced autophagy, and bacillomycin treatment prevented the maturation of autophagogener-induced autophagocytic vacuoles.

Additional discussion

Human genome sequencing has made it possible to predict new genes encoding putative drug targets and to characterize multiple small molecules derived from compound libraries in a high-sample processing and target-derived manner (Drews (2000)). On the other hand, cell-based phenotypic screening of a diverse set of compounds presents a future chemical-genetic approach to identifying small molecules that regulate biological processes in living cells (Haggarty et al. (2000); Stockwell, (2000). )). In the present study, to screen for autophagy inducible small molecules, cell-root, visual, chemical and genetic screening was performed in stable COS7 cell lines expressing EGFP-LC3. The effectiveness of this “phenotype” screening method has led to the discovery of a novel small molecule autophagogenizer (FIG. 1), which has been identified as an autophagy cell death inducer.

Since anticancer drugs generally act as a mechanism for inducing apoptosis, it is not surprising that apoptosis mechanism damage is found in chemotherapy resistant cancer cells (Kaufmann and Vaux (2003); Longley and Johnston (2005)). However, even within apoptotic resistant cells, non-apoptotic cell death is observed in response to chemotherapeutic treatment (Leist and Jaattela (2001); Roninson et al. (2001); Naito et al. (2004)). Many cancer cells are resistant to traditional chemotherapeutic agents as a result of survival through mutations that inactivate apoptosis mechanisms or activate anti-apoptotic pathways. Thus, the characterization of novel anticancer agents that work in an apoptosis-independent manner is deeply related to the novel therapeutic strategies. Targeting autophagy cell death will require identification of novel therapeutic targets and will have a significant impact on attempts to treat diseases associated with abnormally programmed cell death.

In particular, the cytotoxic efficacy of autofaginizer is not limited to HT1080 fibrosarcoma cells. In the panel of eight human cancer cell lines used in the experiments of the present invention, all cells were sensitive to automogonizers in the micromolar range. The inventors have found that the IC ₅₀ from Autopagonizer It was found to be 5 μΜ. Interestingly, the treatment of autophagogener-sensitized cells has undergone progressive saturation. Thus, the saturation of cytosol can be regarded as a characteristic of the killing process induced by the compounds of the present invention.

Several markers were used as well as electron microscopy to observe autophagogenizer-induced vacuoles. In addition, the presence of cytoplasmic double-membrane-bound structures sometimes found in vacuoles may suggest autophagy. Interestingly, structures similar to cytoplasmic organelles have been found in the vacuoles of autophagogenerized cells.

We found that autophagogenizer induced autophagy in HT1080 cells, which was independent of apoptosis and carspace. Indeed, carspace-independent apoptosis or nuclear fragmentation-independent apoptosis have been reported selectively (Kolenko et al., 2000; Ferri and Kroemer, 2000), while the lack of both properties simultaneously is non-apoptosis. It is considered a sign of sexual cell death (Szende et al. (1995); Sasaki et al. (2003); Guo et al. (2003)). In addition, autophagogenizer-induced autophages were inhibited by the known autophagy inhibitors, batillomycin A1.

Interestingly, autophagogenizer-induced autophagy was not inhibited by 3-MA, which is a commonly used reagent for class III PI3K active interference purposes to inhibit autophagy in the sequestration step. . Therefore, cDNA microarray analysis was performed to elucidate the mechanism inherent in the operation of autophagogenizers. Microarray analysis of HT1080 cells confirmed the upregulation of BHMT, and further experiments confirmed the importance of BHMT in autophagogenizer-induced autophagy cell death. In addition, HT1080 cell microarray analysis revealed that autophagy-related molecules, such as beclin-1, are upregulated in HT1080 cells by autophagogenizer treatment, whereas apoptosis-related molecules, such as DAP3 and carspace-9, It was confirmed that the down-regulation. BHMT is an autophagosomal membrane enzyme required for glutathione (GSH) synthesis and is known as a target of 3-MA (Furuya et al. (2001)). 3-MA prevents loss of BHMT activity, resulting in a decrease in intracellular GSH / GSSG ratio. BHMT is an enzyme rich in liver and kidney that catalyzes the transfer of methyl groups from betaine to homocysteine to produce dimethylglycine and methionine (Lee et al. (1992); Garrow (1996)). Cytosol BHMT is ingested during autophagy degeneration and converted to p32, a 32-kDa fragment, which is associated with the fusion of autophagosomes and lysosomes.

In conclusion, the experimental results of the present invention suggested a novel biological function of autofaginizer. The novelty of the autopagonizer structure makes this small molecule an innovative autophagy inducer. Although various autophagy inducers, such as the HDAC inhibitor SAHA and the mTOR inhibitor rapamycin, are already known, autophagoneizers exhibit chemical structures independent of those previously discovered. Identifying the autophagy signal path associated with the novel structure will be the cornerstone for the novel autophagy inducers. Autophagonizer is an inducer of autophagy leading to autophagy cell death and can be used as a therapeutic agent for diseases such as cancer, atherosclerosis and retinal neovascularization. In order to develop an autopagonizer as a therapeutic, it is necessary to clarify its target protein and activity mode. Since the cDNA analysis identified a signal pathway different from rapamycin, the mTOR pathway was excluded from the autophagogenizer signal pathway. Target identification techniques such as phage display or ORFeome for further elucidation of the autopaginizer signal pathways and target proteins are under study, and they will provide insights into unknown mechanisms. In conclusion, Autophagoneizer is a new small molecule that provides a new and unique tool for exploring the exciting field of autophagy biology.

Importance of the present invention

The autophagogenizer of the present invention is a novel small molecule capable of inducing autophagy to cause autophagy cell death. Autophagy inducers having this novel chemical structure were identified by cell-based screening, which showed an increase in autophagy vacuoles in stable cell lines expressing EGFP-LC3. LysoTracker and MDC fluorescence were also significantly increased in autophagogenerized cells. The autophagy efficacy of autophagogenizers inhibited cell proliferation in a large number of cancer cell lines and reduced cell viability resulting in apoptosis-independent autophagy cell death. The mechanism differs from rapamycin because no known autophagy inhibitors have inhibited the autophagy efficacy of autophagogenizers. Thus, autophagogenizers have been identified as autophagy inducers with unique chemical structures and novel modes of activity, which would be a quantum leap in the field of autophagy research.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (10)

(a) a pharmaceutically effective amount of a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof; And (b) a pharmaceutically acceptable carrier, wherein the pharmaceutical composition for preventing or treating cancer comprises:
Formula 1

delete According to claim 1, wherein the cancer is pituitary adenoma, glioma, brain tumor, epiglottis cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, Ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytoma, leukemia, childhood cancer, skin cancer, ovarian cancer and cervical cancer Pharmaceutical compositions.
According to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition, characterized in that up-regulating the expression of beta-homocysteine methyltransferase (BHMT).
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition does not affect apoptosis.
Screening methods for preventing or treating cancer, comprising the following steps:
(a) treating a cell comprising a beta-homocysteine methyltransferase (BHMT) nucleotide sequence with (i) a test substance or (ii) an autophagy inhibitor and a test substance selected from the group consisting of 3-MA, wortmannin and PD98059 ; And
(b) analyzing the expression of the BHMT to screen for cancer prevention or treatment as a step of treating the test substance alone to increase the expression of BHMT to activate autophagy, and simultaneously treated with the autophagy inhibitor and the test substance In one case, the method is characterized in that the screening for cancer prevention or treatment if the autophagy activity is not reduced.
delete According to claim 6, wherein the cancer is pituitary adenoma, glioma, brain tumor, epiglottis cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, Ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytoma, leukemia, childhood cancer, skin cancer, ovarian cancer and cervical cancer Screening method.
7. The method of claim 6, wherein the BHMT nucleotide sequence is a nucleotide sequence of GeneBank BC012616.
The screening method of claim 6, wherein analyzing the expression of BHMT is performed by RT-PCR.

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