WO2019004732A1 - Pharmaceutical composition comprising substance inhibiting enzymatic activity of peroxiredoxin 2 as effective ingredient for treatment of colorectal cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a substance inhibitory of the enzymatic activity of peroxiredoxin 2 as an effective ingredient for treatment of colorectal cancer and, more particularly, to a pharmaceutical composition for treatment of colorectal cancer wherein, on the basis of the mechanism in which peroxiredoxin 2 (Prx II) interacts with tankyrase (TNKS) in APC mutant cells to play a tumor-promoting role in colorectal cancer, the composition exhibits the effect of reducing colorectal tumors by inhibiting the activity of peroxiredoxin 2 to increase the active β-catenin destruction.

Description

A pharmaceutical composition for the treatment of colorectal cancer comprising as an active ingredient a substance which inhibits the enzyme activity of peroxiredoxin 2

The present invention relates to a pharmaceutical composition for the treatment of colorectal cancer comprising as an active ingredient a substance which inhibits the enzyme activity of peroxiredoxin-2.

Cancer is one of the most intractable diseases and is constantly being studied for the cure of patients. In hospitals, techniques such as drug therapy, radiation therapy, gene therapy and the like are used to treat cancer.

Colorectal cancer (CRC) is a common cancer worldwide. In Korea, malignant tumors are followed by gastric cancer, lung cancer, and liver cancer in women, followed by breast cancer and gastric cancer in women. Recently, as the diet has been westernized, the incidence has increased more rapidly. In the last decade, mortality from colon cancer has increased by about 80% and has continued to increase.

About 40-50% of patients with colorectal cancer (CRC) develop recurrence and metastasis. The main cause of death in patients with colorectal cancer and other malignant tumors is not the malignant tumor itself, but the metastasis of malignant tumors. Most metastases are multiple, systemic.

According to large-scale sequencing data, more than 80% of patients with colorectal cancer (CRC) have mutations in the adenomatous polyposis coli (APC). The APC (Adenomatous polyposis coli) protein is an important skeletal protein of the beta-catenin breakdown complex. For this reason, it is known that the active beta-catenin (Wnt-independent) accumulates in APC gene mutant cells to initiate intestinal tumor formation.

Recent studies have shown that intestinal tumorigenesis initiated by APC gene mutation is facilitated by the acquisition or genetic mutation of DNA Glycosylase, an enzyme that plays an important role in repair when the nucleotide base is oxidatively damaged have. This indicates that elevated levels of reactive oxygen species (ROS) are involved in APC mutagenic intestinal tumorigenesis.

Peroxiredoxin (Prx) is known in vivo as a peroxidase of hydrogen peroxide and alkylhydroperoxides. (Chae, H. Z. et al., Proc. Nat. Acad Sci. 91: 7017-7021, 1994). Peroxiredoxin (Prx) is classified as type I to VI Prx isozymes in mammals and is observed in various parts of the tissue (Rhee, SG et al., IUBMB Life 52: 35-41, 2001) .

Peroxiredoxin (Prx) is known to exhibit strong antioxidant activity in cells. Most of the peroxidase isoenzymes except Prx type VI use thioredoxin as electron donor. Therefore, it is known as thioredoxin peroxidase.

The Prx family consists of six isozymes divided into 2-Cys and 1-Cys (Cysteine) subfamilies. The 2-Cys subfamily enzyme is a thioredoxin-dependent peroxidase that is extensively conserved from bacteria to humans. Among 2-Cys Prx, cytoplasmic PrxI and PrxII isozymes are over-expressed in a variety of cancer types and are known to play important regulatory roles in membrane receptor-mediated signal transduction.

The mammalian 2-Cys Prx enzyme is electron-donated from an electron-conveying system consisting of thioredoxin-thioredoxin reductase, and is activated by nicotinamide adenine dinucleotide phosphate (NADPH) In the presence of H ₂ O ₂ (hydrogen peroxide) is reduced to water.

H ₂ O ₂ regulates the reversible oxidation of signaling proteins, including protein kinases or protein tyrosine phosphatases. H ₂ O ₂ thus functions as a potential secondary messenger in proliferating cancer cells. PrxII is known to regulate H ₂ O ₂ locally. Therefore, PrxII, a 2-Cys Prx enzyme, is thought to play a diverse role in the detoxification and signal transduction of intracellular ROS.

Tankyrase (TNKS) is a kind of poly (ADP-ribose) polymerase (Poly (ADP-ribose) polymerase). The activity of Tankyrase (TNKS) in colorectal cancer cells inhibits β-catenin degradation, leading to abnormal cell proliferation. Thus, the inhibition of tannicase is of interest as a target for the treatment of colorectal cancer (CRC), but the direct inhibition of tannicase (TNK) may cause pleiotropic effects or side effects due to a wide range of substrates . Furthermore, the mechanisms controlling the activity of Tankyrase (TNKS) in colon cancer tumors are not well known.

Axis Inhibitor Protein (Axin1) Tumor suppressor is another skeletal protein that constitutes a beta-catenin breakdown complex. The mechanism of Axin1 protein regulation by tannase in APC mutant cells is not clear. In addition, the treatment of colorectal cancer (CRC), which regulates the activity of tannase by controlling the in vivo redox system, has not been attempted so far.

In order to solve the problems of the prior art as described above, the present inventors have found that peroxiredoxin 2 (Prx II), which plays a diverse role in the redox system and signal transduction in APC mutant cells, is expressed by Tankyrase (TNKS) And to provide a pharmaceutical composition for the treatment of colorectal cancer which is capable of inhibiting colon cancer by inhibiting the enzyme activity of peroxiredoxin 2 by such a mechanism.

In order to solve the above problems, one embodiment of the present invention includes a pharmaceutical composition for treating colon cancer, comprising a substance that inhibits the enzyme activity of peroxiredoxin-2 as an effective ingredient.

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance inhibiting the enzyme activity of peroxycorticosin 2 is represented by the following formula (1).

[Chemical Formula 1]

(Wherein R1 is -O-R2 or a compound consisting of a cyclic compound or -H, R2 is a C1 to C8 branched or unbranched alkyl group (Alkyl), or a substituted or unsubstituted benzyl group, -CH = CH2 or an allyl group (Allyl).

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance inhibiting the enzyme activity of peroxiredoxin 2 may include a compound selected from the group consisting of the following compounds-1 to 6 have.

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance inhibiting the enzyme activity of peroxycorticosin 2 is characterized in that β-catenin degradation is increased.

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance that inhibits the enzyme activity of peroxycorticosin 2 is characterized by reducing the degradation of Axin1 by Tankyrase (TNKS).

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance which inhibits the enzyme activity of peroxycorticosin 2 is characterized by increasing oxidative inactivation of Tankyrase (TNKS).

In the pharmaceutical composition for the treatment of colorectal cancer according to the present invention, the oxidative inactivation of the above-mentioned TK occurs in the cytoplasm of APC mutant cells.

In the pharmaceutical composition for treating colorectal cancer according to the present invention, the substance inhibiting the enzyme activity of peroxycorticosin 2 is characterized by inhibiting the interaction of peroxycorticosin 2 and tannase.

The pharmaceutical composition for treating colon cancer according to the present invention is characterized by containing a pharmaceutical effective amount of a substance inhibiting the enzymatic activity of peroxiredoxin-2. The term " pharmaceutically effective amount " of the present invention means an amount sufficient to achieve efficacy or activity of the substance inhibiting the peroxycorticin 2 protein activity.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition are those conventionally used in pharmaceutical preparations such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The nature, form, and advantages of one embodiment of the invention are set forth in the description that follows, and in part will be obvious from the description, or may be learned by practice of the example embodiment thereof.

Other features, forms, and advantages will be readily apparent to those skilled in the art from the following description and claims, and may be learned by practice of the embodiments described below.

According to one embodiment of the present invention, it is possible to provide a composition capable of inhibiting the enzyme activity of peroxiredoxin 2 to regulate the redox system of colon cancer cells, thereby reducing the colonic polyps and treating or preventing colon cancer. According to one embodiment of the present invention, a pharmaceutical composition capable of reducing colonic polyps and treating or preventing colon cancer can be provided by reducing the interaction of PrxII and tannase with the cytoplasm of APC mutant cells. According to one embodiment of the present invention, a method for treating cancer-suppressing colorectal cancer can be provided by controlling intracellular redox system.

FIG. 1 and FIG. 2 show the result of confirming the genotype of a double mutant mouse produced according to an embodiment of the present invention.

Figure 3 shows the results of measurement of peroxycorticosin 2 protein expression in immunofluorescent staining in non-polyp segments and polyp of the small intestine according to one embodiment of the present invention.

Figure 4 is a micrograph of intestinal tissue of a double mutant mouse produced according to one embodiment of the present invention.

FIG. 5 shows the results of measurement of the number of polyps in the intestinal tissues of a double mutant mouse produced according to an embodiment of the present invention.

Figure 6 is a result of staining of the small intestine and colon sections of a double mutant mouse produced according to an embodiment of the present invention with hematoxylin and eosin.

7 is a micrograph of the intestinal tissue of a double mutant mouse produced according to one embodiment of the present invention.

FIG. 8 shows the results of measurement of the number of polyps in the intestinal tissues of a double mutant mouse produced according to an embodiment of the present invention.

9 is a result of immunoblotting using a double-mutant mouse prepared according to an embodiment of the present invention.

FIG. 10 shows the results of measurement of expression of Axin1, β-catenin and β-catenin target genes in the polyp of a double mutant mouse produced according to an embodiment of the present invention.

Fig. 11 shows immunohistochemical image measurement results of Ki-67 in the polyp of a double mutant mouse produced according to an embodiment of the present invention.

FIG. 12 shows the results of measurement of the number of cells proliferating after fluorescent staining with an anti-BrdU antibody in the polyp of a double mutant mouse produced according to an embodiment of the present invention.

FIG. 13 shows the result of immunostaining with anti-BrdU antibody in the polyp of a double mutant mouse produced according to an embodiment of the present invention.

FIG. 14 shows the result of measurement of cell death in a polyp (TUNEL) staining of a polypoid mouse according to an embodiment of the present invention.

Figure 15 shows the results of immunoblot (IB) measurements of beta catenin infecting cells with a set of siRNA specific for PrxI and PrxII for 48 hours according to one embodiment of the present invention.

Figure 16 shows the results of immunoblot (IB) measurement of beta-catenin by infecting cells with different concentrations of PrxII-1 siRNA for 48 hours according to one embodiment of the present invention.

17 is a graph showing the results of immunization with PrxII-1 siRNA in various APC mutant colorectal cancer cell lines for 48 hours according to one embodiment of the present invention and immunization against beta-catenin and active beta-catenin Blowing (IB) measurements.

FIGS. 18 and 19 are graphs showing the expression of PrxII active form (WT) and inactive form (CS or C172S) after infection with PrxII-1 siRNA in APC mutant colorectal cancer cell line for 48 hours according to an embodiment of the present invention (IB) measurements of β-catenin and active β-catenin in retrovirus-infected cells.

FIG. 20 shows the results of measurement of Prx II-dependent cell H ₂ O ₂ levels in APC mutant colorectal cancer cells according to an embodiment of the present invention.

FIGS. 21-23 are graphs showing the inhibition of proteasome and GSK3 beta (glycogen synthase kinase-3 beta) inhibitors (25 μM MG132 and lactacystin) in the control and PrxII-deficient SW480 and HT29 colon cancer cell lines according to an embodiment of the present invention 2.5 [mu] M BIO and SB216763) for 1 hour and measuring the beta-catenin and active beta-catenin expression levels, indicating that beta-catenin destruction complex is involved Results.

24 is a graph showing that PrxII deficiency does not affect the activation of GSK3? In APC mutant colorectal cancer cells according to an embodiment of the present invention.

25 is a graph showing that PrxII deficiency in the HEK293 cell line according to an embodiment of the present invention does not affect normal Wnt3A signal stimulation.

26 shows the results of measurement of β-catenin / TCF transcription activity according to an embodiment of the present invention.

27 to 32 are results of analysis of genes differentially expressed in HT29 and SW480 cells by PrxII deficiency according to an embodiment of the present invention

FIG. 33 shows the results of transfecting H. 29 and SW480 cells according to an embodiment of the present invention with a control group or PrxII-1 siRNA and immunoblotting against active beta-catenin and Axin1.

Figure 34 shows the results of immunoblotting of all of the complex binding proteins obtained by transfecting SW480 cells with the control group or PrxII-1 siRNA and isolating Axin-1 by immunoprecipitation (IP) according to an embodiment of the present invention It is the result that PrxII deficiency greatly increases the complex.

FIG. 35 shows the results of transfection of HΔ29 cells according to an embodiment of the present invention with control or Axin-1 / Axin-2 siRNAs and immunoblotting against active beta-catenin and Axin1.

FIG. 36 shows the results of immunohistochemical staining for transfection of ΗT29 and SW480 cells with a control or PrxII-1 siRNA according to one embodiment of the present invention, immunoprecipitation of Axin-1 followed by immunoblotting for ubiquitination and poly-ADP-risose Results.

FIG. 37 shows the result of transfecting APC wild-type colorectal cancer cell line RKO and APC mutant cell lines, Ht29 and SW480 cells with a control group or PrxII-1 siRNA and performing a colony line assay according to an embodiment of the present invention.

38 is a graph showing the results of transfecting the APC mutant cell lines, ΗT29 and SW480 cells, with a control group or PrxII-1 siRNA, followed by expressing the activated beta-catenin (S37A), followed by a colony line assay to be.

39 and 40 are then HT29 and RKO cells, the control group or to transfect siRNA into PrxII-1 or H ₂ O ₂ by immunoprecipitation (IP) and then treated with a TNKS1 according to one embodiment of the present invention ADP- risose polymerase (PARP) activity.

Figure 41 shows the results of transfection with a control or PrxII-1 siRNA in various APC wild-type and mutant colorectal cancer cell lines according to an embodiment of the present invention, and the expression of TNKS1 and Axin1 and TRF1 as substrates of the enzyme by immunoblotting to be.

FIG. 42 shows the results of pretreatment of TNKS1 inhibitor (XAV939) for 1 hour in various APC wild-type and mutant colorectal cancer cell lines according to an embodiment of the present invention, and then the expression of TNKS1 and the enzymes Axin1 and TRF1 were measured by immunoblotting This is a result.

43 shows the results of measuring the degree of cell proliferation after pretreatment of the TNKS1 inhibitor (XAV939) for 1 hour in various APC mutant colorectal cancer cell lines according to an embodiment of the present invention.

FIG. 44 shows the results of measurement of intracellular H ₂ O ₂ level after deficiency of APC gene expression using siRNA in RKO colorectal cancer cells according to an embodiment of the present invention.

FIG. 45 shows the results of immunoblot measurement of levels of beta-catenin, Axin1, and TNKS1 according to an embodiment of the present invention.

Figure 46 shows the results of measurement of ADP-ribose polymerase activity of TNKS1 according to one embodiment of the present invention.

FIG. 47 shows the results of measurement of ADP-ribose polymerase activity of TNKS1 after immunization with anti-TNKS antibody by expressing various TNKS1 cysteine mutant enzymes in cells according to an embodiment of the present invention.

48 shows the results of expression and purification of a recombinant protein in E. coli cells against the PARP domain of TNKS1 according to an embodiment of the present invention.

Figure 49 shows the results of measurement of the extraction of Zinc ions by H ₂ O ₂ treatment in the purified recombinant TNKS1-PARD protein according to an embodiment of the present invention.

FIG. 50 is a result of immunoblotting the cells according to an embodiment of the present invention with respect to a protein expressed after immunoprecipitation with an anti-TNKS antibody.

Figure 51 is a co-immunoprecipitation (co-IP) result for detecting binding between TNKS and PrxII protein according to one embodiment of the present invention.

Figures 52 and 53 are results of in situ proximity ligation assays according to one embodiment of the present invention.

Figures 54 and 55 are the results of analyzing the mutual binding of each other using truncation mutations or single residue mutations of TNKS and Myc-PrxII, respectively, according to an embodiment of the present invention.

Figures 56 and 57 are the results of measurement of intracellular expression and activity of peroxidase of wild-type and G116V mutants of PrxII according to an embodiment of the present invention.

Figure 58 shows the results of performing PARP activity analysis of TNKS1 in cells expressing PrxII wild type and G116V mutant according to an embodiment of the present invention.

Figure 59 shows the results of performing a colony forming assay on cells expressing PrxII wild type and G116V mutant according to an embodiment of the present invention.

Figures 60 and 61 show the expression levels of the PrxI and Prxll genes of colon cancer patients (CRC, n = 155) from healthy individuals and the Cancer Genome Atlas (TCGA) code database according to one embodiment of the present invention .

Figure 62 shows PrxII immunostaining results in the colon arrangement of healthy human and colorectal cancer patients according to one embodiment of the present invention.

FIG. 63 shows the results of inhibition of the activity of Compound-6 (conoidine A) against PRXI and PrxII according to an embodiment of the present invention.

FIG. 64 is a result of performing a colony forming assay on a colon cancer cell line pretreated with Compound-6 (a compound of the present invention) (Compound 6) according to an embodiment of the present invention.

65 to 66 are graphs showing the results of xenografting a compound cancer cell line HT29 treated with Compound-6 (chondroitin A) according to an embodiment of the present invention and then measuring the growth of the tumor by in vivo luminescence imaging and weighing This is a result.

67 is a schematic diagram showing the effect of inhibiting PrxII enzyme activity according to an embodiment of the present invention to inhibit colon cancer.

68 shows the results of measuring the expression level of PrxII gene in patients with colorectal cancer having APC WT and APC mutations according to an embodiment of the present invention.

69 shows results of peroxidase activity measured by reacting PrxII with Compound-1 in vitro in accordance with an embodiment of the present invention.

FIG. 70 shows the results of peroxidase activity measured by reacting PrxII with Compound-2 in vitro in accordance with an embodiment of the present invention.

71 shows the results of peroxidase activity measured by reacting PrxII with Compound-3 in vitro in accordance with an embodiment of the present invention.

72 is a result of peroxidase activity measured by reacting PrxII with Compound-4 in vitro in accordance with an embodiment of the present invention.

FIG. 73 shows the results of peroxidase activity measured by reacting PrxII with Compound-5 in vitro in accordance with an embodiment of the present invention.

FIGS. 74 and 75 show results of an experiment for culturing RKO cell colonies of the compound I-1 and the compound-5 according to the present invention.

FIGS. 76 and 77 show results of culturing HT29 cell colonies of the compound 1 with compound 1 and compound 5 according to an embodiment of the present invention. FIG.

Brief Description of the Drawings The advantages and features of the present invention, and how to accomplish it, will become apparent with reference to the embodiments described hereinafter with reference to the accompanying drawings. However, these examples are for further illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Throughout the specification, when a section includes a constituent element, it is understood that it can include other constituent elements, not excluding other constituent elements unless specifically stated otherwise.

As used herein, the terms Compound-1 to Compound-6 refer to the substances listed in Table 1 below.

≪ Example 1 > Cell culture

All CRC and HEK293 cells were provided from the American Type Culture Collection (Manassas, VA, USA). SW480, DLD1, CoLo205, Colo741 and SW620 cells were subcultured in RPMI 1840 medium supplemented with 10% fetal bovine serum.

HEK293 and RKO cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS. HT29 cells were cultured in McCoy 's 5A medium supplemented with 10% FBS. Mycoplasma contamination was periodically tested in cell culture supernatants using mycoplasma detection kit (Biotool, USA).

Example 2: Preparation of double-mutant mice

To examine the CRC-specific function of PrxII in vivo, double-mutant mice were made by crossing PrxI +/- and PrxII +/- mice with APCMin / + mice.

PrxI +/- and PrxII +/- C57BL / 6 mice were crossed with APC Min / + mice according to C57BL / 6 background (Jackson Laboratory, Bar Harbor, USA) and cultivated and maintained in an aseptic facility to obtain APCMin / +; PrxI + / + APCMin / +; PrxI +/-, APCMin / +; PrxI - / -, APCMin / +; PrxII + / +, APCMin / +; A complex mutant showing the genotype of PrxII +/- and APCMin / +; Prx II - / - was constructed.

The littermates were genotyped by genomic PCR of specific primers and rat tail DNA. Literate refers to the cubs born on a ship. :

All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University and complied with the ARRIVE guidelines. Animal experiments were carried out in a double-blind fashion by separating animal sarcoma and tissue analysis.

In the tumor xenograft model, mice were anesthetized by inhalation of isoflurane gas (N2O: O2 / 70%: 30%) and HT29-luc2 cells (2.5x10 5 cells) suspended in 200 μl of PBS Lt; / RTI > Intraperitoneal administration of compounds 1 to 6 (286 [mu] M in DMSO) was started 6 days after cell injection and repeated every 3 days.

Bioluminescent imaging was performed with IVIS Lumina Series III (Perkin Elmer). For each imaging session, luciferin in PBS (Luciferin 150 mg / kg body weight) was administered intraperitoneally according to the manufacturer's protocol. Up to four animals were maintained in an integral anesthetic manifold instrument and imaged 10 min after luciferin injection. The IVIS imaging system collects photographic images and quantitative bioluminescent signals of mice and then overlaps them.

Example 3 Double-mutant mouse genotype determination

The genotypes of the double mutation-induced mice prepared in the above Example were determined by genomic PCR at 4 weeks, and the genotypes of the double mutant mice produced by the above-mentioned Examples were determined in FIGS. 1 and 2 .

In FIGS. 1 to 3, it can be confirmed that the double mutant mouse produced by the above example generates multiple intestinal neoplasia (Min) by mutation of APC (adenomatous polyposis coli) cleavage. The products for the APC gene cleaved in Figures 1 and 2 are indicated by an asterisk.

Although the APC mutation is heterozygous, it is known that polymorphonuclear polyposis is caused by the disappearance of residual APC wild type (WT), and the resulting adenomatous polyposis is the result of human colon cancer It is known to contain a truncated APC wild type (WT) copy similar to the tumor.

The primers used to determine the genotype are as follows.

APC ^Min

(wild type) 5'-GCCATCCCTTCACGTTAG-3 ',

(mutant) 5'-TTCTGAGAAAGACAGAAGTTA-3 '

(common) 5'-TTCCACTTTGGCATAAGGC-3 '

PrxI

forward 5'-CTGGAAACCTGGCAGTGATA-3 ',

reverse, 5'-CTGTGACTGATAGAAGATTGGT-3 '),

PrxII

forward, 5'-GATGATCTCCGTGGGGCAAACAAA-3 ',

reverse, 5'-ATGGCCTCCGGCAACGCGCAAATC-3 ',

Neo cassette

forward, 5'-GCTTGGGTGGAGAGGCTATTCG-3 ',

reverse, 5'-GTAAAGCACGAGGAAGCGGTCAGC-3 '.

Example 4: Observation of adenomatous polyps in double-mutant mice

The small intestine and colon were excised from the main mouse, and the adenomatous polyp of the intestine was separated and observed using a stereoscopic microscope, and is shown in Fig. 4

The number of polyps having a diameter of more than 0.3 mm in the small intestine and colon of the mouse was measured and shown in Fig. FIG APC ^{Min / +,} as shown in 5; PrxII ^{- / -} number of the small intestine and colon on average greater than the visible diameter 0.3 mm in polyps of mice APC ^{Min / +;} PrxII ^{+ / +} and APC ^{Min / +;} PrxII ^{+ / -} about 50% as compared to the number of mouse polyps.

^{APC Min / +; PrxII - /} - mice (median survival = 241 days) ^{APC Min / +; PrxII + /} + ( median survival = 146 days) and APC ^{Min / +;} PrxII ^+/- (median survival = 152 days ) Survived much longer than.

The histological examination of the small intestine and the large intestine of the mice was carried out, and the results are shown in Fig. As shown in FIG. 6, Prx II deficiency did not change the villi structure but decreased frequency and size of adenomatous polyps.

The adenomatous polyps of the intestine for PrxI-deficient mice were observed using a stereomicroscope and the number of them was counted, as shown in FIGS. 7 and 8. FIG. The average number of intestinal polyps in APC ^{Min / +} ; PrxI ^{- / -} was equal to the number of APC ^{Min / +} ; PrxI ^{+ / +} and APC ^{Min / +} ; PrxI ^+/- polyps.

These results suggest that PrxII promotes intestinal tumors induced by APC mutations in vivo, whereas PrxI is independent of APC mutation induced intestinal tumors.

Example 5 Expression of beta-catenin

The amount of β-catenin and its gene expression in polyps isolated from APC ^{Min / +} ; PrxII ^{+ / +} and APC ^{Min / +} ; PrxII ^{- / -} was measured by immunoblotting and the results are shown in FIG.

As shown in FIG. 9, the expression levels of β-catenin and its transcriptional targets c-Myc and cyclin D1 in APC ^{Min / +} ; PrxII ^{- / -} tumors are shown in APC ^{Min / +} ; PrxII ^{+ / +} Lt; RTI ID = 0.0 > tumor < / RTI > However, expression level of Axin1, an important scaffold protein in the β-catenin destruction complex, was increased in APC ^{Min / +} ; PrxII ^{- / -} mice in inverse proportion to the tumor.

As shown in FIG. 10, since the amount of mRNA expression of β-catenin and Axin1 was not changed between APC ^{Min / +} ; PrxII ^{+ / +} and APC ^{Min / +} ; PrxII ^{- / -} , PrxII was expressed in vivo It can be seen that the expression of Axin1 and beta-catenin is regulated at the protein level.

Example 6: Measurement of whether beta-catenin target gene is involved in the proliferation and survival of CRC cells

To determine whether the β-catenin target gene is involved in the proliferation and survival of CRC cells, proliferating and dead cells were counted.

As seen in the Ki-67 expression and BrdU incorporation assays of Figures 11 and 12, the percentage of proliferating cells in the APC ^{Min / +} ; PrxII ^{+ / +} and APC ^{Min / +} ; PrxII ^{- / -} This was similar.

Also, in Fig. 13, BrdU incorporation assays showed that PrxII deficiency had no effect on the proliferation and migration of intestinal epithelial cells of the scrotum.

In contrast, the number of dead cells measured by TUNEL staining in polyps of APC ^{Min / +} ; PrxII ^{- / -} in FIG. 14 was significantly higher than the number of dead cells measured by TUNEL staining in APC ^{Min / +} ; PrxII ^{+ / +} Respectively. These results suggest that PrxII promotes the survival of tumor cells in the adenomatous polyposis induced by APC mutation.

<Example 7> Analysis of regulation mechanism of β-catenin expression by PrxII

The mechanism of β-catenin expression regulation by PrxII in human CRC cells overexpressing PrxII was analyzed.

As shown in FIG. 15, in the APC-mutant CRC cells SW480 and HT29 cells, the decrease in expression of PrxII during siRNA analysis markedly decreased the expression of endogenous beta-catenin, and the cut mutant APC protein Lt; / RTI &gt; However, PrxI deficiency did not alter beta-catenin expression in both SW480 and HT29 cells.

In addition, as shown in FIG. 16, the decrease in β-catenin expression is proportional to the degree of PrxII deficiency, which suggests that strict knockdown of PrxII is important for decreasing the β-catenin level Able to know.

As shown in Figure 17, PrxII deficiency was also associated with total beta-catenin and active beta-catenin (non-phosphorylated forms) in SWC220, DLD-1 and CoLo205, other APC- mutant CRC cells The amount of expression was decreased.

To exclude off-target effects of PrxII siRNA, PrxII was expressed by transfecting HT29 cells with the siRNA-resistant form of PrxII.

As shown in FIGS. 18 and 19, the PrxII wild type (WT) completely restored the total beta-catenin and active beta-catenin levels compared to control cells transfected with siRNA This confirms the specific role of PrxII in regulating β-catenin expression.

Conversely, expression of PrxII in peroxidase-inactive mutants C172S and C51 / 172S failed to restore the level of beta-catenin expression, suggesting that peroxidase activity of PrxII is active in CRC cells It is necessary to maintain the active beta-catenin level. In Figure 20, PrxII deficiency increased intracellular H ₂ O ₂ levels in HT29 and SW480 cells.

We found that PrxII deficiency did not alter β-catenin mRNA levels, suggesting that β-catenin (β-catenin) was induced by casein kinase-1 and GSK-3β (glycogen synthase kinase-3β) ) Was sequenced by a canonical destruction complex, which indicates the phosphorylation of the β-catenin protein.

Phosphorylated beta-catenin is ubiquitinated by an enzyme called β-TrCP (Beta-transducin repeats-containing proteins) or E3 ubiquitin E3 ligase and degraded by proteasome , We examined whether the β-catenin destruction complex is involved in PrxII-dependent regulation of β-catenin expression.

21-23, glycogen synthase kinase-3β (GSK3β) inhibitors increased active β-catenin expression, suggesting that constitutively-active GSK3β (glycogen synthase kinase-3β) .

However, as shown in FIG. 24, it can be seen that PrxII deletion is not likely to stimulate GSK3β (glycogen synthase kinase-3β) activation from the tyrosine phosphorylation level of GSK3β (glycogen synthase kinase-3β), which is an index of kinase activation .

25, PrxII deficiency did not affect Wnt-induced beta-catenin stabilization, and deregulated beta-catenin signaling showed that PrxII selectively inhibited Wnt-induced beta-catenin stabilization I confirmed that I participated.

As a result of reduced beta-catenin expression in Figure 26, PrxII deficiency induced a significant decrease in TCF-dependent reporter expression.

<Example 8> Beta-catenin / TCF-dependent transcriptional regulation by PrxII deficiency

β-catenin / TCF-dependent transcription was examined by mRNA sequencing in HT29 cells by mRNA sequencing, and the results are shown in FIGS. 27, 28, 29 and 30.

As shown in FIG. 31, in particular, 12 out of the beta-catenin target genes expressed in CRC cells containing a major beta-catenin target gene such as CCND1, AXIN2 and BIRC5 by PrxII deletion Was down regulated (FDR <0.05).

Other metastatic and cell cycle-promoting genes such as S100A4, MMP7, ID2 and PTTG1 were also downregulated by PrxII deficiency.

In addition to HT29 cells, PrxII deficiency downregulated 13 β-catenin target genes in SW480 cells (FDR <0.05), and in FIG. 32, four of these genes (CCND1, S100A4, ID2, EDN1) .

Down-regulation of several other genes in SW480 cells implies that they can be mediated by a secondary effect of β-catenin-related transcriptional complexes or β-catenin reduction.

These results suggest that PrxII deficiency accelerates the degradation of transcriptionally active β-catenin through CRC cell-destructive complexes.

&Lt; Example 9 > Beta-catenin due to PrxII deficiency in CRC cells [ of  Wnt-independent and Axin1-dependent breakdown analysis

Axin1, another scaffold protein in the APC mutation, is known to play an important role in beta-catenin destruction. Axin1 overexpression in APC-mutant CRC cells is well known to induce β-catenin degradation.

In FIG. 9, it has been confirmed that Axin1 expression is increased in the intestinal polyp of the PrxII gene-deleted rat.

The amount of destructive complexes associated with Axin1 and Axin1 was measured in CRC cells. Immunoblot analysis in FIG. 33 showed that PrxII deficiency increased endogenous Axin1 protein expression in both HT29 and SW480 CRC cells, indicating an inverse correlation with active beta-catenin expression.

In the co-immunoprecipitation experiment of Fig. 34, it can be seen that the PrxII deletion increased the expression of Axin1-related destructive complexes. When the glycogen synthase kinase-3β inhibitor, BIO, was treated, the phospho-β-catenin disappeared from the complex.

In Fig. 35, the knockdown of Axin1 / 2 restored the active beta-catenin level of PrxII deficient cells to the level of control cells.

These results indicate that Axin in PrxII-deficient CRC cells forms a functional burst complex that regulates β-catenin degradation.

Since Axin is degraded by poly (ADP-ribose) polymerisation (PARPylation) and subsequent ubiquitination, the status of Axin1 was analyzed in HT29 and SW480 cells.

Indeed, treatment of the proteasome inhibitor MG132 induces the accumulation of poly ADP-lysed PARSylated and ubiquitinated Axin1 in control cells, indicating that Axin1 is continuously degraded.

Conversely, as shown in Fig. 36, PrxII deficiency inhibited poly ADP-lyvosylation / ubiquitination of Axin1 without affecting intracellular total ubiquitination.

Example 10: Measurement of colony forming ability of PrxII-deficient CRC cells

To evaluate the biological significance of the PrxII-dependent regulatory mechanism of the Axin1 / beta-catenin pathway, colony forming ability in CRC cells was investigated.

Analysis of in vitro colony forming ability shows that PrxII deficiency in HT29 and SW480 cells, which are not RKO cells expressing APC WT, sufficiently inhibits colony formation in FIG.

In FIG. 38, it can be seen that the expression of the active beta-catenin S37A mutant almost completely restores the ability of the APC mutant CRC cells to be damaged by the PrxII deletion.

From this, it can be seen that the PrxII deficiency can induce the β-catenin degradation by Axin1, thereby sufficiently reversing the carcinogenic phenotype of the APC mutation.

Example 11: Regulatory analysis of TNKS-Axin1 signaling by PrxII deficiency

TNKS is the only poly ADP ribocylating enzyme for Axin proteins. It was confirmed by in vitro PARP analysis whether PrxII is essential for TNKS activity.

As shown in FIG. 39, PrxII deficiency induced severe damage of TNKS activity in APC-mutant HT29 and SW480 cells, but not in APC-functioning RKO cells. In contrast, H ₂ O ₂ treatment inhibited TNKS activity in both SW480 and RKO cells, as shown in FIG.

From the above results, it can be seen that the exogenous H ₂ O ₂ directly inactivates the TNKS activity regardless of the APC mutation.

Because TNKS is known to undergo auto-poly ADP lysylation (auto PARsylation) and degradation, TNK expression levels were measured with the substrate protein in CRC cell panels.

As shown in FIG. 41, in all the APC-mutant CRC cells subjected to the experiment, the PrxII deletion increased the expression level of TNKS and Axin1, but the expression level did not increase in the APC-functioning cells including RKO and Colo741 cells.

More importantly, PrxII deficiency did not affect the expression level of TRF1 (telomeric repeat-binding factor 1), another TNKS substrate essential for telomerase regulation in the nucleus.

In Figures 42 and 43, by contrast, direct inhibition of TNKS using a specific inhibitor, XAV939, increased the expression of TNKS and substrates Axin1 and TRF1 in all CRC cells and consequently inhibited the proliferation of HT29 and SW480 cells Respectively.

APC knockdown was performed on RKO cells to confirm a direct correlation between APC and PrxII function in CRC cells.

As shown in FIGS. 44, 45 and 46, the APC knockdown definitely induces a marked increase in the expression of H ₂ O ₂ and β-catenin in the cells, and consequently, the H ₂ O ₂ -dependent fire of TNKS1 Acceleration of activation. In addition, concurrent deficiency of APC and PrxII increased TNKS and Axin1 protein expression with a decrease in β-catenin expression.

Since PrxII is a cytoplasmic peroxidase, PrxII suggests that it can selectively protect intracellular TNKS from oxidative stress induced by APC mutation or loss.

Example 12 Confirmation of Inactivation Mechanism of PARP Activity of H2O2-Mediated TNKS

To identify the mechanism of inactivation of the PARP activity of H ₂ O ₂ -mediated TNKS, the oxidation-sensitive Cys residues in the PARP catalytic domain of TNKS were screened.

The peptide sequences of the various PARP domains were aligned to reveal five Cys residues containing three zinc binding motifs and found to be unique in the TNKS isoform between the PARP lines.

The mutation of three zinc-coordinated Cys residues (C1234, C1242 and C1245) of the five Cys residues found in the TNKS1 mutated Cys residues to Ser and examined its PARP activity revealed that PARP activity Resulting in complete loss of

The recombinant TNKS1 PARP domain (amino acids 1023-1327) was prepared to test if zinc binding motifs were unstable under oxidative conditions. The TNKS1 PARP domain exhibited intact poly ADP lysylating activity and was completely inactivated by incubation with H ₂ O ₂ as shown in FIG.

The zinc binding protein is produced by cysteine oxidation, and the released zinc ions are measured with a spectrophotometer using 4- (2-pyridylazo) resorcinol. .

Fig. 49 shows the result of measurement by a spectrophotometer by the above method. In Figure 44B, it can be seen that the H ₂ O ₂ treatment induced almost complete release of zinc ions, with more than 90% of the TNKS1 PARP protein ultimately losing zinc ions.

These results indicate that the zinc binding motif of TNKS is essential for PARP activity and H ₂ O ₂ -induced oxidation of the Cys residue induces the release of zinc ions from the PARP domain.

From these results, it can be seen that the oxidation-reduction of TANKS is controlled by PrxII-regulated H ₂ O ₂ .

<Example 13> Selective binding between PrxII and TNKS

The interaction between PrxII and TNKS was examined to demonstrate how PrxII protects TNKS from H ₂ O ₂ mediated inactivation.

In Figure 50, which shows the results of co-immunoprecipitation (IP) experiments, it can be seen that endogenous TNKS interacts with Prx II only in APC-mutant HT29 and SW480 cells, not in APC-functional RKO cells.

In contrast, TNKS did not interact with PrxI in APC-mutant HT29 and SW480 cells, confirming the specific role of PrxII in the TNKS / Axin1 / β-catenin pathway.

To characterize the direct interaction between TNKS1 and PrxII, two proteins were overexpressed in human embryonic kidney cells HEK293 as non-CRC cells. As a result, it can be seen that TNKS and PrxII directly interact in the co-IP experiment as shown in FIG.

More specifically, the in situ proximity ligation assay (in situ PLA) in Figures 52 and 53 demonstrates that direct interaction between TNKS and PrxII occurs in the cytoplasm of HT29 and SW480 cells that are not RKO cells Visualized. In Figure 52 and Figure 53, the red fluorescence signal indicative of the interaction of the two proteins almost completely disappeared by PrxII deficiency.

Therefore, it can be seen that the redox-shielded TNKS by PrxII is very specific by protein-protein interaction and is dependent on the APC mutation.

TNKS and PrxII mutagenesis were performed to analyze the molecular interaction map. The IP experiment in Figure 54 showed that PrxII interacted with an ankyrin repeat cluster (ARC) 4/5 domain of TNKS when truncated TNKS mutants were expressed with PrxII, It does not overlap Axin1 binding to the 2/3 domain.

The TNKS ARC domain recognizes the consensus sequence RXXPXG in the client protein, and the Gly residue at position 6 plays a crucial role in direct binding.

In PrxII, a similar hexapeptide sequence was searched and three potential common regions were found and a Gly-to-Val mutation was introduced. In Fig. 55 showing the results of the Co-IP experiment, it can be confirmed that only the G116V mutation among the three mutant sites completely destroys the binding of PrxII and TNKS1.

56 and 57, the PrxII WT and G116V mutants exhibited the same level of expression and peroxidase activity. However, as shown in FIG. 53, the PrxII G116V mutant did not prevent the inhibition of TNKS activity by H ₂ O ₂ , The PrxII WT was completely blocked.

The Prx-SO2 _/ 3 blot in lane 2 and lane 3 of Figure 58 also shows that the endogenous 2-Cys Prx is completely peroxidized during H ₂ O ₂ treatment whereas the exogenous Prx II (PrxII-Myc) with C-terminal Myc tag Is resistant to peroxidation.

Actually, as a result of analysis of the activity of in vitro 2-Cys Prx using a recombinant enzyme, as shown in FIG. 58, unlike wild-type PrxII enzyme, PrxII-Myc enzyme showed strong peroxidase activity without any signs of peroxidation.

Since it is known that the C-terminal modification of the PrxII protein confers resistance to peroxidation, the addition of the Myc tag at the C-terminus predicts that similar structural changes may occur in PrxII, which translates into a peroxidation-resistant form.

In addition, a colony formation assay was performed to determine the biological significance of the PrxII-TANKS interaction.

In FIG. 59, colony formation of APC-mutant SW480 cells inhibited by PrxII deficiency was completely restored by ectopic expression of PrxII WT, but the G116V mutant was not completely recovered

From these results, it can be seen that binding PrxII can prevent the oxidative inactivation of tannase by removing H ₂ O ₂ from the tannase, which is important for the growth of APC-mutated CRC cells.

Example 14 Expression of PrxII in Human Colon Cancer Tissue

As shown in FIG. 60, PrxII expression was significantly higher in tumor samples of patients with colorectal adenocarcinoma than in normal colon tissues, and the expression of PrxI, the closest homologous protein, . In Figure 61, increased PrxII expression was observed at all tumor stages.

As shown in FIG. 62, in the immunohistochemistry using the CRC tissue array, the PrxII level was about two times higher in the CRC tissue than in the normal tissue. From these results, it can be seen that certain PrxII induction can be a prerequisite for CRC expansion.

Example 15 Regulation of PrxII Activity by Compound 6 (Conoidine A)

On the basis of the above analysis, it was found that Compound 6, called Conoidin A, which is known to covalently bind to PrxII in order to evaluate the possibility of treating human CRC by inhibiting PrxII Cell permeable compounds were tested.

In the in vitro Prx assay of Figure 63, compound 6 conoid A suppressed human Prx II activity to about 85% (214.9 ± 24.3 nmol min ^{-1 for} control and 38.8 ± 17.1 nmol min ^{-1 for} conoidin A treatment) , And PrxI activity was suppressed to about 50%.

In contrast, the initial rate of PrxI activity was not affected by compound 6 conoid A (263.7 ± 45.4 nmol min -1 for the control, 241.7 ± 21 nmol min -1 for the conoididin A)

Example 16 Analysis of Colony Formation by Treatment with Compound 6 (Conoidine A)

In FIG. 64, colony formation analysis showed that treatment with Compound 6 (conoid A) sufficiently inhibited proliferation of HT29 and SW480 cells that are not RKO cells. Lt; RTI ID = 0.0 &gt; PrxII &lt; / RTI &gt; inhibition to selectively target human APC mutant CRC cells.

When compound 6 (chondroitin A) was intraperitoneally injected into rats bearing HT29-derived tumor xenografts, treatment with compound 6 (chondroitin A), as shown in Figures 65 and 66, Significantly delayed growth.

From this, it can be seen that PrxII can be a new target treatment for human CRC as shown in the schematic diagram of FIG. 67, and a compound that inhibits PrxII such as Compound 6 (conoid A) can be a new therapeutic drug for human CRC Able to know.

Hereinafter, the synthesis method of the compound-1 to the compound-5 will be described in the synthesis examples. Compound-6 is conoidine A and is widely sold, such as Candia Thamtech Company Limited, Shanghai YuLue Chemical Co., Ltd.

Synthesis Example 1 Synthesis of 2,3-bis (bromomethyl) -6-methoxyquinoxaline 1,4-dioxide (2,3-bis (bromomethyl) -6-methoxyquinoxaline 1,4- -1)>

Step 1

4-Methoxy-2-nitroaniline (1.01 g, 5.89 mmol) was added to a previously prepared 20% KOH / ethanol solution (35 ml) and stirred. Approximately 30 ml of a 12% NaOCl solution is added dropwise in an ice water bath, then the temperature is raised to room temperature and stirred until the starting material disappeared.

Filter the resulting solids and wash with cold ethanol. The yellow solid thus obtained is subjected to recrystallization using a water: ethanol = 1: 3 solution (0.65 g, 66%).

¹ H-NMR (400 MHz, CDCl 3)? 7.47-6.40 (m, 3H), and 3.90 (s, 3H).

Step 2

6-Methoxybenzo [c] [1,2,5] oxadiazole 1-oxide (1.00 g, 6.02 mmol) was dissolved in triethyl And stirred with trimethylamine (18.06 mmol). Pyrrolidine (0.78 g, 10.83 mmol) and methylethylketone (0.65 g, 9.03 mmol) were added dropwise in an ice bath.

After about 1 hour after the temperature rise, the reaction was completed (TLC analysis, n-hexane: EtOAc = 2: 1). The reaction solution was filtered and then filtered through cold ethanol to obtain a brown solid (1.06 g, 80%). At this stage, the next step was carried out without further purification.

J = 9.0 Hz, 3.0 Hz, ¹ H-NMR (400 MHz, DMSO-d 6)? 8.36 (d, J = 9.6 Hz, 1H) 1H), 3.97 (s, 3H), 2.59 (s, 3H), and 2.56 (s, 3H).

Step 3

6-methoxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.54 mmol) was dissolved in 1,4-dioxane (1,4 -dioxide, bromine (12.71 mmol) was added dropwise.

The reaction solution was heated to 90 ° C and reacted for about 2 hours until the spot of starting material disappeared. The progress of the reaction was judged by TLC analysis (n-Hexane: EtOAc = 1: 2).

After completion of the reaction, NaHCO ₃ aqueous solution was added thereto and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated to obtain a mixture. This product was subjected to column chromatography using MPLC (n-Hexane: EtOAc = 3: 1) to obtain Compound-1 (1.15 g, 65%).

^{1 H-NMR (400 MHz,} DMSO-d 6) (d, J = 7.4 Hz, 2.2 Hz), 5.06 (s, 4H), 8.08 (d, J = and 4.01 (s, 3H); ¹³ C-NMR (100 MHz, DMSO-d ₆ )? 162.9, 124.4, 122.2, 99.5, 57.0, 23.5, and 23.4; HRMS (ESI): m / z 377.9038 [M + H] + (calcd for C 11 H 10 Br 2 N 2 O 3 + 378.9170)

Synthesis Example 2 Synthesis of 2,3-bis (bromomethyl) -6-ethoxyquinoxaline 1,4-dioxide (2,3-bis (bromomethyl) -6-ethoxyquinoxaline 1,4-dioxide) -2)>

Step 1

4-Amino-3-nitrophenol (5.00 g, 32.44 mmol) and bromoethane (5.30 g, 48.66 mmol) were stirred in DMF solvent and potassium carbonate (6.73 g, 48.66 mmol).

Then, the temperature of the reaction vessel was raised to 90 ° C and the reaction was observed for about 3 hours (TLC analysis condition, n-hexane: EtOAc = 3: 1) until the starting material disappeared.

When the reaction is completed, the reaction solution is added to the ice water after cooling at room temperature. The resulting solid was filtered to give a red solid in the form of a mixture. This was purified through a column (n-Hexane: EtOAc = 10: 1) to finally identify 4-ethoxy-2-nitroaniline (4.25 g, 72%).

^{1 H-NMR (400MHz, CDCl3} ) δ 7.55 (bs, 1H), 7.07 (d, J = 8Hz, 1H), 6.77 (d, J = 8Hz, 1H), 5.89 (bs, 2H), 4.01 (m, 2H), and 1.41 (t, J = 6.0 Hz, 3H).

Step 2

4-Ethoxy-2-nitroaniline (2.00 g, 10.98 mmol) was added to a previously prepared 20% KOH / ethanol solution (60 ml) and stirred.

About 50 ml of a 12% NaOCl solution is added dropwise in an ice water bath, then the temperature is raised to room temperature and stirred until the starting material disappeared. Filter the resulting solids and wash with cold ethanol. The yellow solid thus obtained is subjected to recrystallization (1.85 g, 94%) using a water: ethanol = 1: 3 solution.

^{1 H-NMR (400 MHz,} CDCl3) δ 7.49 - 6.34 (m, 3H), 4.08 (m, 2H), and 1.48 (t, J = 8.0 Hz, 3H)

Step 3

6-Ethoxybenzo [c] [1,2,5] oxadiazole 1-oxide (1.50 g, 8.33 mmol) was dissolved in triethyl And stirred with trimethylamine (2.52 g, 24.98 mmol).

Pyrrolidine (1.08 g, 14.99 mmol) and methylethylketone (0.90 g, 12.49 mmol) were added dropwise in an ice bath.

After about 1 hour after the temperature rise, the reaction was completed (TLC analysis, n-hexane: EtOAc = 2: 1). The reaction solution was filtered and then washed with cold ethanol to obtain a brown solid (1.11 g, 57%). At this stage, the next step was carried out without further purification.

^{1 H-NMR (400 MHz,} DMSO- d6) δ 8.51 (d, J = 9.6 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.38 (dd, J = 9.4, 2.6 Hz, 1H) , 4.24 (m, 2H), 2.74 (s, 3H), 2.71 (s, 3H), and 1.51 (t, J = 6.8 Hz, 3H).

Step 4

6-ethoxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.27 mmol) was dissolved in 1,4-dioxane (1,4 -dioxide, bromine (12.71 mmol) was added dropwise.

The reaction solution was heated to 90 ° C and reacted for about 2 hours until the spot of starting material disappeared. The progress of the reaction was judged by TLC analysis (n-Hexane: EtOAc = 1: 2). After completion of the reaction, NaHCO3 aqueous solution was added thereto and extracted with ethyl acetate.

The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to obtain a product in a mixed state. This product was subjected to column chromatography using MPLC (n-Hexane: EtOAc = 3: 1) to obtain Compound-2 (0.98 g, 61%).

^{1 H-NMR (400 MHz,} DMSO-d6) δ 8.52 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.42 (dd, J = 9.6, 2.8 Hz), 4.90 (s, 4H), 4.24 (m, 2H), and 1.52 (t, J = 7.2 Hz, 3H); ¹³ C-NMR (100 MHz, DMSO-d ₆ )? 163.2, 122.5, 122.1, 101.2, 58.0, 23.9 and 13.9; HRMS (ESI): m / z 391.9194 [M + H] + (calcd for C 12 H 12 Br 2 N 2 O3 + 392.9155)

SYNTHESIS EXAMPLE 3 Synthesis of 2,3-bis (bromomethyl) -6-isopropoxyquinoxaline 1,4-dioxide (2,3-bis (bromomethyl) -6-isopropoxyquinoxaline 1,4- -3)>

Step 1

4-Amino-3-nitrophenol (5.00 g, 32.44 mmol) and 2-bromopropane (5.98 g, 48.66 mmol) were stirred in DMF solvent, Potassium carbonate (6.73 g, 48.66 mmol) is added in small portions.

Then, the temperature of the reaction vessel was raised to 90 ° C to observe the reaction for about 5 hours until the starting material disappeared (TLC analysis condition, n-hexane: EtOAc = 3: 1).

When the reaction is completed, the reaction solution is added to the ice water after cooling at room temperature. The mixture was then extracted with diethyl ether until the spot disappeared in the aqueous layer. The organic layer was washed with brine, dried over MgSO4 and concentrated to obtain a red solid.

This was purified through a column (n-Hexane: EtOAc = 10: 1) to finally identify 4-isopropoxy-2-nitroaniline (5.90 g, 93%).

^{1 H-NMR (400MHz, CDCl3} ) δ7.51 (bs, 1H), 7.04 (d, J = 8.0Hz, 1H), 6.75 (d, J = 8.0Hz, 1H), 5.88 (bs, 2H), 4.46 (m, 1H), and 1.33 (d, J = 6.0 Hz, 6H).

Step 2

4-Isopropoxy-2-nitroaniline (2.00 g, 10.19 mmol) was added to a previously prepared 20% KOH / ethanol solution (60 ml) and stirred.

About 50 ml of NaOCl 12% solution is added dropwise in an ice water bath, then the temperature is raised to room temperature and stirred until the starting material disappeared. Filter the resulting solids and wash with cold ethanol. The yellow solid thus obtained is subjected to recrystallization (1.93 g, 97%) using a water: ethanol = 1: 3 solution.

¹ H-NMR (400 MHz, CDCl 3)? 7.48-6.34 (m, 3H), 4.60 (m, 1H), and 1.41 (d, J = 6.0 Hz, 6H).

Step 3

6-Isopropoxybenzo [c] [1,2,5] oxadiazole 1-oxide (1.50 g, 7.72 mmol) was dissolved in tri And stirred with trimethylamine (2.34 g, 23.17 mmol).

Pyrrolidine (1.00 g, 13.90 mmol) and methylethylketone (0.84 g, 11.59 mmol) were added dropwise in an ice bath. After about 1 hour after the temperature rise, the reaction was completed (TLC analysis, n-hexane: EtOAc = 2: 1). The reaction solution was filtered and washed with cold ethanol to obtain a brown solid (1.49 g, 78%). At this stage, the next step was carried out without further purification.

^{1 H-NMR (400 MHz,} DMSO- d6) δ 8.50 (d, J = 9.6 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.34 (dd, J = 9.4 Hz, 2.6 Hz, 1H ) 4.81 (s, 1H), 2.73 (s, 3H), 2.71 (s, 3H), and 1.44 (d, J = 6.0 Hz, 3H).

Step 4

6-isoproxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.03 mmol) was dissolved in 1,4-dioxane (1,4 -dioxide, bromine (12.71 mmol) was added dropwise.

The reaction solution was heated to 90 ° C and reacted for about 2 hours until the spot of starting material disappeared. The progress of the reaction was judged by TLC analysis (n-Hexane: EtOAc = 1: 2).

After completion of the reaction, NaHCO ₃ aqueous solution was added thereto and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated to obtain a mixture. The product was subjected to column chromatography using MPLC (n-Hexane: EtOAc = 3: 1) to obtain Compound-3 (0.69 g, 42%).

^{1 H-NMR (400 MHz,} DMSO-d 6) δ 8.52 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.42 (dd, J = 9.6, 2.8 Hz), 4.90 (s, 4 H), 4.24 (m, 2 H), and 1.52 (t, J = 7.2 Hz, 3 H); ¹³ C-NMR (100 MHz, DMSO-d ₆ )? 162.9, 122.70, 105.7, 73.0, 23.9 and 21.8; HRMS (ESI): m / z 405.9351 [M + H] + (calcd for C 13 H 14 Br 2 N 2 O3 + 406.9458)

Synthesis Example 4: Synthesis of 6- (allyloxy) -2,3-bis (bromomethyl) quinoxaline 1,4-dioxide ) Synthesis (Compound-4) &gt;

Step 1

4-Amino-3-nitrophenol (5.00 g, 32.44 mmol) and allyl bromide (5.89 g, 48.66 mmol) were stirred in DMF solvent and potassium carbonate (6.73 g, 48.66 mmol).

Then, the temperature of the reaction vessel was raised to 90 ° C and the reaction was observed for about 3 hours (TLC analysis condition, n-hexane: EtOAc = 3: 1) until the starting material disappeared.

When the reaction is completed, the reaction solution is added to the ice water after cooling at room temperature. Then, diethyl ether was added until the spot disappears in the water layer. The organic layer was washed with brine, dried over MgSO4 and concentrated to obtain a red solid.

This was purified through column (n-Hexane: EtOAc = 10: 1) to give 4- (allyloxy) -2-nitroaniline (4,44 g, 71% Respectively.

^{1 H-NMR (400 MHz,} CDCl3) δ7.52 (bs, 1H), 7.00 (d, J = 7.6Hz, 1H), 6.73 (d, J = 8.0Hz, 1H), 6.06 (m, 1H), 5.60 (bs, 2H), 5.47 (d, J = 16.0 Hz, 1H), 5.34 (d, J = 10.4 Hz, 1H), and 4.70 (d, J = 5.2 Hz, 2H).

Step 2:

4-Allyloxy-2-nitroaniline (2.00 g, 10.30 mmol) was added to a previously prepared 20% KOH / ethanol solution (60 ml) and stirred.

About 50 ml of NaOCl 12% solution is added dropwise in an ice water bath, then the temperature is raised to room temperature and stirred until the starting material disappeared. Filter the resulting solids and wash with cold ethanol. The yellow solid thus obtained is subjected to recrystallization (1.96 g, 99%) using a water: ethanol = 1: 3 solution.

¹ H-NMR (400 MHz, CDCl 3)? 7.76-6.69 (m, 3H), 6.06 (m, 1H).

Step 3:

6- (Allyloxy) benzo [c] [1,2,5] oxadiazole 1-oxide (1.50 g, 7.81 mmol) are stirred together with trimethylamine (2.36 g, 23.42 mmol).

Pyrrolidine (1.01 g, 14.05 mmol) and methylethylketone (0.84 g, 11.71 mmol) were added dropwise in an ice bath. After about 1 hour after the temperature rise, the reaction was completed (TLC analysis, n-hexane: EtOAc = 2: 1). The reaction solution was filtered and washed with cold ethanol to obtain a brown solid (0.96 g, 50%). At this stage, the next step was carried out without further purification.

¹ H-NMR (400 MHz, DMSO- d 6)? 8.38 (d, J = 9.6 Hz, 1H), 7.82 , 6.10 (m, IH), 5.57 (dd, J = 17.2, 1.6 Hz, IH), 5.34 (dd, J = 10.6, 1.6 Hz, IH), 2.59 (s, 3H), 2.57 (s, 3H).

Step 4:

6- (allyloxy) -2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.06 mmol) was dissolved in 1,4-dioxane (1,4-dioxide), bromine (12.71 mmol) was added dropwise.

The reaction solution was heated to 90 ° C and reacted for about 2 hours until the spot of starting material disappeared. The progress of the reaction was judged by TLC analysis (n-Hexane: EtOAc = 1: 2). After completion of the reaction, NaHCO ₃ aqueous solution was added thereto and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated to obtain a mixture.

This product was subjected to column chromatography using MPLC (n-Hexane: EtOAc = 3: 1) to obtain Compound-4 (0.72 g, 44%).

^{1 H-NMR (400 MHz,} DMSO-d 6) δ 8.25 (d, J = 9.6 Hz, 1H), 7.90 (m, 2H), 5.87 (m, 1H) 5.23 ~ 5.17 (m, 2H), 4.92 ( s, 4H), and 4.61 (d, J = 4.8 Hz, 2H); ^{13 C-NMR (100MHz, DMSO} -d 6) δ 167.0, 134.5, 122.3, 122,1, 117.5, 105.0, 70.1, and 23.9 HRMS (ESI): m / z 403.9194 [M + H] + (calcd for C ₁₃ H ₁₂ Br ₂ N ₂ O 3 + 404.9279)

Synthesis Example 5: Preparation of 2,3-bis (bromomethyl) -6- (prop-2-yloxy) -ynyloxy) quinoxaline 1,4-dioxide (Compound-5))>

Step 1:

4-Amino-3-nitrophenol (5.00 g, 32.44 mmol) and propargyl bromide (5.79 g, 48.66 mmol) were stirred in a DMF solvent and potassium carbonate ) (6.73 g, 48.66 mmol). Then, the temperature of the reaction vessel was raised to 90 ° C and the reaction was observed for about 3 hours (TLC analysis condition, n-hexane: EtOAc = 3: 1) until the starting material disappeared.

When the reaction is completed, the reaction solution is added to the ice water after cooling at room temperature. The mixture was then extracted with diethyl ether until the spot disappeared in the aqueous layer. The organic layer was washed with brine, dried over MgSO4 and concentrated to obtain a red solid.

This was obtained via a column (n-Hexane: EtOAc = 10: 1) to give 2-nitro-4- (prop-2-yn- 1-yloxy) yloxy) aniline (3.95 g, 63%) was finally confirmed.

^{1 H-NMR (400 MHz,} CDCl3) δ7.69 (bs, 1H), 7.13 (d, J = 8.0Hz, 1H), 6.78 (d, J = 8.0Hz, 1H), 5.91 (bs, 2H), 4.67 (d, J = 4.0 Hz, 2H), and 2.55 (t, J = 2.0 Hz, 1H).

Step 2:

2-nitro-4- (prop-2-yn-1-yloxy) aniline (2.00 g, 10.41 mmol) Add 20% KOH / ethanol solution (60 ml) and stir. About 50 ml of NaOCl 12% solution is added dropwise in an ice water bath, then the temperature is raised to room temperature and stirred until the starting material disappeared.

Filter the resulting solids and wash with cold ethanol. The yellow solid thus obtained is subjected to recrystallization (1.96 g, 99%) using a water: ethanol = 1: 3 solution.

¹ H-NMR (400 MHz, CDCl 3)? 7.81-6.81 (m, 3H), 4.98 (d, J = 2.0 Hz, 2H), and 3.34 (s, 1H).

Step 3:

6-Prop-2-yn-1-yloxy benzo [c] [1,2,5] oxadiazole 1-oxide [ 1,2,5] oxadiazole 1-oxide (1.50 g, 7.89 mmol) were stirred together with trimethylamine (2.39 g, 23.66 mmol).

Pyrrolidine (1.02 g, 14.20 mmol) and methylethylketone (0.85 g, 11.83 mmol) were added dropwise in an ice bath. After about 1 hour after the temperature rise, the reaction was completed (TLC analysis, n-hexane: EtOAc = 2: 1). The reaction solution was filtered and then washed with cold ethanol to obtain a brown solid (0.96 g, 51%). At this stage, the next step was carried out without further purification.

^{1 H-NMR (400 MHz,} DMSO- d6) δ 8.38 (d, J = 9.6 Hz, 1H), 7.92 (d, J = 2.4, 1H), 7.51 (dd, J = 9.4, 2.6 Hz, 1H), (D, J = 2.4 Hz, 2H), 3.71 (t, J = 2.4 Hz, 1H), 2.59 (s, 1H), 2.57 (s, 1H).

Step 4:

2,3-dimethyl-6- (prop-2-ynyloxy) quinoxaline 1,4-dioxide (1.00 g, 4.09 mmol) was dissolved in 1,4- (dioxa) 1,4-dioxide and bromine (12.71 mmol) was added dropwise.

The reaction solution was heated to 90 ° C and reacted for about 2 hours until the spot of starting material disappeared. The progress of the reaction was judged by TLC analysis (n-Hexane: EtOAc = 1: 2). After completion of the reaction, NaHCO ₃ aqueous solution was added thereto and extracted with ethyl acetate.

The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated to obtain a mixture. This product was subjected to column chromatography using MPLC (n-Hexane: EtOAc = 3: 1) to obtain Compound-5 (0.68 g, 41%).

^{1 H-NMR (400 MHz,} DMSO-d 6) δ 8.34 (d, J = 9.6 Hz, 1H), 8.06 (m, 2H); 5.00 (s, 4H), 4.86 (d, J = 2.4 Hz, 2H) and 3.51 (t, J = 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ )? 164.8, 122.2, 120.6, 106.3, 80.0, 78.8, 58.8 and 23.1; HRMS (ESI): m / z 401.9038 [M + H] + (calcd for C 13 H 10 Br 2 N 2 O3 + 402.9465)

<Example 17> Control of PrxII activity by Compound-1 to Compound-5

To evaluate the possibility of treating human CRC by inhibiting PrxII, the cytopathic compounds of Compound-1 to Compound-5 were tested.

In the graphs of FIG. 72 to FIG. 73, it can be confirmed that Compound-1 to Compound-5 synthesized according to one embodiment of the present invention significantly inhibits PrxII activity.

Example 18 Analysis of Colony Formation by Compound-1 to Compound-5

74 shows RKO cells treated with Compound-1 to Compound-5 and measuring the number of colonies. 75 shows the result of colony formation analysis in which RKO cells were treated with Compound-1 through Compound-5 and conoidin A (Compound-6).

76 shows the number of colonies after treatment with Compound-1 to Compound-5 in HT29 cells. 77 shows the result of analysis of colony formation in which HT29 cells were treated with Compound-1 to Compound-5 and conoidin A (Compound-6).

5 inhibited the proliferation of HT29 cells that were not RKO cells after treatment with Compound-1 to Compound-5. From this, PrxII can be a new target therapy for human CRC, and compounds that inhibit PrxII, such as conoid A (compound-6) and compound-1 through compound-5, can be new therapeutic agents for human CRC .

&Lt; Reference Example 1 > The Cancer Genome Atlas (TCGA analysis)

Expression of PrxI and PrxII in colorectal cancer and normal colon tissue samples was assessed using microarray data from the Cancer Genome Atlas (TCGA) project (https://tcga-data.nci.nih.gov) Respectively.

To illustrate, mRNA expression data was generated using an Agilent G4502A microarray platform and processed and standardized as previously described. For analysis of gene expression data, 155 colon cancer tissue samples and 26 normal colon tissue samples were included.

&Lt; Reference Example 2 > Immunoblotting and immunoprecipitation [

The lumen of the intestine was washed with ice-cold phosphate buffered saline (PBS) using a flat needle syringe and longitudinally incised. The polyp-free section of the intestine was resected for immunoblot analysis with the polyp.

Tissues were suspended in HEPES-buffered saline containing 10% glycerol, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 5 mM Na3VO4, 5 mM NaF, 1 mM AEBSF, aprotinin, 5 μg / ml, leupeptin Homogenized using a Dounce homogenizer.

The cultured cells were rinsed once with ice cold PBS and resuspended in 20 mM HEPES pH 7.0, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 5 mM Na3 VO4, 5 mM NaF, 1 mM AEBSF, aprotinin (5 μg / ml) and leupeptin (5 μg / ml).

Tissue homogenates and cell lysates were centrifuged at 15,000 x g for 15 minutes and protein concentrations were examined by Bradford assay (Pierce). Protein samples were mixed with SDS sample buffer and boiled for 5 minutes.

The protein was separated by SDS-PAGE and electroblotted for 1 hour to transfer to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) or 5% dried skim milk for 2 hours in Tris buffered saline containing 0.05% (v / v) Tween-20 (TBST) And incubated with appropriate primary antibodies in blocking buffer.

Then, after 3 washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) in blocking buffer. The immune-reactive bands were detected with a chemiluminescence kit (AbFrontier, Korea) and quantified with a LAS-3000 imaging system (Fuji Film, Japan).

If necessary, strip the membrane by shaking in 67 mM Tris (pH 6.7), 2% SDS and 100 mM β-mercaptoethanol for 60 min at 37 ° C and re-assay with appropriate normal antibodies.

For immunoprecipitation, the purified cell lysate (0.5-1 mg protein) was pre-removed with 30 μl protein-A / G Sepharose 4 Fast Flow beads (Amersham Biosciences) for 1 hour.

The supernatant was incubated overnight with 3 μg of the appropriate antibody and then precipitated again by mixing with 30 μl of protein-A / G beads at 4 ° C for 3 hours.

The beads were then washed three times with 1 ml of lysis buffer followed by in vitro PARP analysis or immunoblotting.

REFERENCE EXAMPLE 3 Beta-catenin / TCF transcription reporter assay (β-catenin / TCF transcription reporter assay)

SW480 cells were plated in 12-well plates and transfected with pTOPflash or pFOPflash plasmids. To normalize the transfection efficiency, cells were transfected with the pRL-TK Renilla Luciferase control plasmid.

After transfection, cells were cultured in complete medium for 24 hours and then lysed in reporter lysis buffer. Luciferase activity was measured three times with the Dual-Luciferase reporter assay (Promega).

Data were reported as fold induction as compared to the control siRNA group after normalization of transfection efficiency. Fold induction is the ratio of experimental activity to control activity.

&Lt; Referential Example 4 > RNA Sequence analysis (RNA-Seq analysis)

Four samples of siRNA-transfected HT29 cells were prepared for high-throughput mRNA sequencing. One μg of RNA was extracted from each sample and an mRNA library (~300 bp insert size) was generated using the TruSeq RNA Library Preparation kit v2 (Illumina).

Paired-end transcriptome sequencing (101 bp read length) was performed on an Illumina HiSeq 2500. The number of reads for each sample ranges from 69.4 million to 74.8 million. Sequencing data was deposited with the GEO database (accession number is GSE81429).

After standard quality checks and trimming with FastQC and Fastx-toolkits, RNA sequence data was aligned with the human genome (hg19 of UCSC) using MapSplice v2.1.7. The mapping rate of reads ranged from 96.5 to 96.8%, and RSG v1.2.12 was used to estimate the abundance of the transcriptome of refGene mRNA.

Differentially expressed genes (DEG) were identified using DESeq2 with an FDR cutoff of 0.05

<Reference Example 5> In vitro PARP assay (in vitro PARP assay)

Immunocomplex-bound beads were incubated in 40 μl of assay buffer (50 mM Tris-HCl pH 8.0, 4 mM MgCl 2) containing 4 μCi of γ-32 [P] -NAD + at 25 ° C for 30 min Lt; / RTI &gt; The reaction is stopped by the addition of 2x SDS sample buffer. The sample is boiled and separated using SDS denaturing gel (SDS denaturing gel).

The gel was vacuum dried and irradiated on an imaging plate. The radioactivity recorded on the plate was read and quantified by Fujifilm Bio-imaging Analyzer System (BAS) -3000.

&Lt; Reference Example 6 > Plasmid construction and site-directed mutagenesis [

A plasmid containing the full-length complementary DNA of human turkey-1 (TNKS1) was purchased from Open Biosystems (mRNA accession number, BC098394). The entire sequence of the Tankyrase-1 was sequenced by forward and reverse primers (underlined in the NotI and BglII sites, respectively)

5'-ATAAGAATGCGGCCGCGGCGGCGTCGCGTCGCTC-3 'and

5'-GAAGATCTCTAGGTCTTCTGCTCTG-3 '), and inserted into p3 × FLAG CMV9 vector to generate flag-tagged TNKS1.

For the domain mapping experiments, various tankyrase-1 fragments were PCR amplified and subcloned into the p3x FLAG CMV9 vector using the following forward and reverse primers:

Amino acid residues 1-158, 5'-ATAAGAATGCGGCCGCGGCGGCGTCGCGTCGCTC-3 'and

5'-GAAGATCTCTAGGCCGCCTCGGGGCTCTC-3 ';

Amino acid residues 158-595, 5'-ATAAGAATGCGGCCGCCGGAGTTAGCAGCACAGCAC-3 'and

5'-GAAGATCTCTACAAAGCAGTCTGACCAAGGG-3 ';

Amino acid residues 596-1022, 5'-ATAAGAATGCGGCCGCGCATAGAGCCGCCCTAGCAGG-3 'and

5'-GAAGATCTCTATCCTTCCTTCCTTTCTGTTCC-3 ';

Amino acid residues 1023-1327, 5'-ATAAGAATGCGGCCGCAGAAGTTGCTGGTCTTGAC-3 'and

5'-GAAGATCTCTAGGTCTTCTGCTCTG-3 '

E. coli expression plasmid for GST-TNKS1 (1023-1327) was donated from Chang-Woo Lee (Sungkyunkwan University School of Medicine).

Retroviral vectors (pQ-CXIX) expressing Myc-tagged siRNA-resistant PrxII WT, C172S single mutants and C51 / 172S double mutants were prepared as described above. Site-directed mutagenesis for amino acid substitution was performed using the QuikChange kit (Stratagene).

Double strand primers for Cys-Ser substitution in tankyrase-1 are as follows:

In the case of the C1163S mutant,

(sense) 5'-GTTGAGGGAGCGGTTCTCCCACCGACAGAAGGAAG-3 ';

In the case of the C1234S mutant,

(sense) 5'-GGAGGAGGAACAGGCTCCCCTACACACAAGGAC-3 ';

For the C1242S mutant,

(sense) 5'-CACAAGGACAGGTCATCCTATATATGTCACAGAC-3 ';

For the C1245S mutant,

(sense) 5'-CAGGTCATGCTATATATCTCACAGACAAATGCTCTTC-3 ';

In the case of the C1252S mutant,

(sense) 5'-GACAAATGCTCTTCTCTAGAGTGACCCTTGGG-3 '

The double strand primers for Gly-Val substitution for human PrxII are as follows:

For the G9V mutant, (sense) 5'-GCGCGCATCGTAAAGCCAGCCCCTG-3 ';

For the G23V mutant, (sense) 5'-GCGGTGGTTGATGTCGCCTTCAAAG-3 ';

For the G116V mutant, (sense) 5'-CTGAGGATTACGTCGTGCTGAAAAC-3 '.

The retroviral pQ vector expressing the beta-catenin S37A variant was constructed by PCR subcloning from the previously described pBI-EGFP-beta-catenin (S37A) structure. All structures and mutations were confirmed by nucleotide sequencing.

<Reference Example 7> Zinc determination

Zinc ions are observed in aqueous solution using 4- (2-pyridylazo) resorcinol (PAR).

The glutathione S-transferase (GST) -fused TNKS1 PARP (1023-1327) protein was expressed in Escherichia coli grown in LB medium supplemented with 100 μM ZnCl2 and incubated in Glutathione Sepharose 4B high-speed flow according to the manufacturer's protocol (GE Healthcare Life Sciences) And purified by affinity chromatography using beads (Glutathione Sepharose 4B Fast Flow beads).

The purity (> 99.5%) of the GST-TNKS1 PARP protein is determined by the concentration method and dialyzed extensively in Chelex100 treatment buffer containing 25 mM HEPES (pH 7.0) and 2 mM DTT to remove unbound zinc ions.

In the GST-TANK1 the PARP protein 40mM HEPES containing 0.1mM PAR (pH 7.0) 200μl reaction buffer for 30 min and incubated with 500μM H ₂ O _2. The formation of the PAR2-Zn2 + complex was monitored at 500 nm with a UV / VIS spectrophotometer (Agilent).

The total zinc content of the purified GST-TNKS1 PARP protein used in the assay was determined by adding 0.5 mM p-chloromercuribenzoic acid to the reaction mixture.

<Reference Example 8> Peroxiredoxin assay (Peroxiredoxin assay)

The peroxidase assay was performed in a 200 μl reaction mixture containing 250 μM NADPH, 1.5 μM yeast TR, 3 μM yeast Trx, recombinant human Prx (PrxI, 4.6 μM, PrxII, 16.4 μm) and 1 mM EDTA 50mM HEPES (pH 7.0) that was performed at 200μM H ₂ O _2.

The mixture (minus H ₂ O ₂ ) was preliminarily incubated for 5 minutes in the presence or absence of compounds 1 to 6 (100 μM), and then H ₂ O ₂ was added to initiate the reaction. NADPH oxidation was monitored for 5 min at 30 ° C with a decrease in absorbance around 340 nm on an Agilent UV8453 spectrophotometer (Hewlett Packard, USA). The initial reaction rate was calculated using the linear portion of the curve and expressed as the amount of oxidized NADPH per minute.

<Reference Example 9> Histology, immunohistochemical staining and immunofluorescence staining (Histology, immunohistochemistry, and immunofluorescence staining)

12-week-old male rats were anesthetized by inhalation of isoflurane gas (N2O: O2 / 70%: 30%) and perfused the heart with heparinized saline containing 3.7% formaldehyde transcardiac perfusion-fixation).

Then the bowel was resected and cut into two parts, small intestine and colon, both opened vertically and folded outward. The folded sheet was paraffin embedded and segmented by a rotary microtome (Leica RM2255).

Three serial tissue sections (10 μm thick) were stained with hematoxylin and eosin (HE). The folded sheet was immediately buried in the OCT medium and frozen in dry ice. A cryotome of frozen tissue was used to cut the tissue into 10μm sections.

Cryotome is a microtome that deals with frozen tissue. A microtome is a machine that cuts a specimen into pieces of uniform thickness to make a specimen for microscopic observation. Placed on a Superfrost Plus slide (Surgipath Medical Inc. UK), dried at room temperature and held at -80 ° C until thawed for immunostaining.

For immunohistochemistry, paraffin sections were waxed off with xylene and rehydrated in ethanol. Antigen retrieval was then performed by boiling the sections in sodium citrate buffer (pH 6.0).

Tissue sections were incubated with anti-Ki-67 antibody (1: 200 dilution) and affinity-purified anti-PrxII antibody (1: 500 dilution) for 48 h at 4 ° C. After washing three times with PBS, the sections were incubated with a peroxidase-conjugated secondary antibody and incubated with a 3,3'-diaminobenzidine (DAB) substrate solution Lt; / RTI &gt;

The nuclei were further stained with hematoxylin. DAB staining images were obtained and quantified using HistoFAXS tissue analysis system (HistoFAXS Tissue Analysis System, TissueGnostics, USA). For immunofluorescence staining, paraffin or frozen sections were blocked with 5% normal rabbit serum (Vector Laboratories) in PBST (0.3% Triton X-100 in PBS) for 1 hour at room temperature.

The sections were incubated overnight at 4 ° C with primary antibody (1: 500 dilution for anti-PrxII antibody and 1: 100 dilution for anti-BrdU antibody). After washing the PBST several times, the samples were incubated with Alexa Fluor 568 conjugated donkey anti-rabbit IgG antibody for 2 hours at room temperature.

The sections were counterstained with (4 ', 6-diamidino-2-phenylindole, DAPI, Sigma-Aldrich) for 30 min and Vectashield mounting medium Respectively.

Fluorescence images were acquired in three random fields per tissue section at 100x magnification using an LSM 51 Meta confocal microscope equipped with argon and a helium-neon laser (Carl Zeiss, Germany).

&Lt; Reference Example 10 > Statistical analysis [

Unless otherwise specified, the data were analyzed using the Student's t-test for comparison between the two groups, or multiple groups (SPSS 12.0K for Windows, SPSS, Illinois, USA) to determine statistical significance Analysis of Variance (ANOVA) was performed using Tukey's honestly significant difference, or Tukey-HSD, or Tukey test. P <0.05 was considered statistically significant.

<Reference Example 11> Data availability

RNA sequencing data supporting this study are deposited in the GEO (Gene Expression Omnibus DB, GEO DB) database (accession number: GSE81429).

<Reagent>

Antibacterials (mouse monoclonal, B-5-1-2, 1: 8000, T5168) and anti-FLAG antibodies (mouse monoclonal, M2, 1: 1000, F3165) were purchased from Sigma-Aldrich.

β-catenin (rabbit monoclonal, 6B3, 1: 1000, 9582), Axin1 (rabbit monoclonal, C76H11, 1: 1000, 2087), pS33 / 37pT41- rabbit monoclonal, 76G6, 1: 1000, 2151), GSK3β (rabbit monoclonal, 27C10, 1: 1000, 9315), β- actin (rabbit monoclonal, 13E5, 1000, 2922) were purchased from Cell Signaling Technology.

 rabbit polyclonal, 1: 1000, sc-135653), c-Myc (rabbit polyclonal, 1: 1000, sc-788), mouse monoclonal, P4D1, 1: 1000, sc-8017, pY279 / Tankyrase-1/2 (rabbit polyclonal, 1: 1000, H-350) was purchased from Santa Cruz Biotechnology.

anti-Myc (agmouse monoclonal, 9E10, 1: 1000, 05-419) and anti-APC (mouse monoclonal, FE9, , ABC202) antibody was purchased from Millipore.

 Alexa Fluor 568-conjugated donkey anti-rabbit IgG (1: 200, A-21206) and anti-β-TrCP (mouse monoclonal, 1B1D2, 1: 1000, 37-3400) antibodies were purchased from Invitrogen Respectively.

Anti-PAR antibody (rabbit polyclonal, 1: 2000, 551813), which detects the poly (ADP-ribose) chain, was purchased from BD Bioscience.

Anti-Ki-67 antibody (rabbit monoclonal, SP6, 1: 200, MA5-14520) was purchased from Thermo Fisher Scientific.

Produced rabbit polyclonal antibodies to PrxI (1: 3000), PrxII (1: 3000) and Prx-SO2 / 3 (1: 1000) as previously described.

Rabbit anti-PrxII antisera were affinity-purified with recombinant PrxII protein and agarose gel beads and used for immunofluorescence and proximity ligation assays (PLA).

Wnt3a was purchased from R & D Biosystems.

DuoLink in situ fluorescence reagents were purchased from Sigma-Aldrich.

TissueFocus colon cancer microarray was purchased from OriGene Technologies (Rockville, USA)

While the present invention has been described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is understandable. Accordingly, the true scope of the present invention should be determined by the appended claims.

According to one embodiment of the present invention, it is possible to provide a composition capable of inhibiting the enzyme activity of peroxiredoxin 2 to regulate the redox system of colon cancer cells, thereby reducing the colonic polyps and treating or preventing colon cancer.

According to one embodiment of the present invention, a pharmaceutical composition capable of reducing colonic polyps and treating or preventing colon cancer can be provided by reducing the interaction of PrxII and tannase with the cytoplasm of APC mutant cells.

Claims (8)

1. A pharmaceutical composition for the treatment of colorectal cancer comprising as an active ingredient a substance which inhibits the enzyme activity of peroxiredoxin 2
2. The method according to claim 1,

   Wherein the substance inhibiting the enzymatic activity of peroxycorticosin 2 is at least one selected from substances represented by the following formula 1:

   [Chemical Formula 1]

   

   In Formula 1,

   R1 is a compound consisting of -O-R2 or a cyclic compound or -H,

   R2 is any one or more selected from the group consisting of C1 to C8, branched or unbranched alkyl groups (Alkyl), or substituted or unsubstituted aromatic or nonaromatic cyclic compounds, -CH = CH2 or allyl groups .
3. The pharmaceutical composition for the treatment of colorectal cancer according to claim 1, wherein the substance inhibiting the enzyme activity of peroxycorticosin 2 is at least one selected from the group consisting of the following compounds-1 to 6

   
4. The method according to claim 1,

   Wherein the substance inhibiting the enzymatic activity of peroxycorticosin 2 increases beta-catenin degradation.
5. The method according to claim 1,

   Wherein the substance that inhibits the enzymatic activity of peroxycorticosin 2 reduces the degradation of Axin1 by a &lt; RTI ID = 0.0 &gt;
6. The method according to claim 1,

   Wherein the substance that inhibits the enzymatic activity of peroxycorticosin 2 increases oxidative inactivation of Tankyrase (TNKS).
7. 6. The method of claim 5,

   Wherein the oxidative inactivation of said Tankyrase (TNKS) occurs in the cytoplasm of APC mutant cells.
8. The method according to claim 1,

   The substance that inhibits the enzymatic activity of peroxycorticosin 2 is a composition that reduces the interaction between peroxiredoxin 2 and Tankyrase (TNKS) in the cytoplasm of APC mutant cells

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