Method for determining sensitivity to simultaneous inhibitor against PARP and Tankyrase

Abstract

The present invention relates to a method for determining sensitivity to a simultaneous inhibitor against poly ADP ribose polymerase (PARP) and Tankyrase. According to the present invention, a colorectal treatment effect can be maximized by sorting patients having sensitivity to the simultaneous inhibitor against PARP and Tankyrase.

Claims

1. A method for treating a human colorectal cancer patient having a normal type of the p53 gene and a mutant type of the LIG4 gene in a biological sample from the patient, comprising administering to said patient a therapeutically effective amount of [(dimethylamino)methyl]-10-ethoxy-1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridin-5(6H)-one or 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile.

2. The method according to claim 1, wherein the mutant type of the LIG4 gene is one or more sequence variants selected from the group consisting of a substitution of cytosine to thymine at position 8, a substitution of cytosine to thymine at position 26, a substitution of guanine to adenine at position 833 and a substitution of thymine to cytosine at position 1704 of SEQ ID NO: 2.

3. The method according to claim 1, wherein the mutant type of the LIG4 gene is one or more sequence variants selected from the group consisting of a substitution of alanine to valine at position 3, a substitution of threonine to isoleucine at position 9, a substitution of arginine to histidine at position 278 and a substitution of thymine of aspartic acid (GAT) to cytosine at position 568 of SEQ ID NO: 3.

4. The method according to claim 1, further comprising: (a) isolating or having isolated cells from the biological sample; (b) isolating or having isolated nucleic acid molecules of the cells of the step (a); and (c) detecting that the genotype of the p53 gene is a normal type and the genotype of the LIG4 gene is a mutant type, by using a primer or a probe that specifically binds to a nucleotide sequence encoding a p53 gene and a primer or a probe that specifically binds to a nucleotide sequence encoding an LIG4 (DNA ligase 4).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the results of analysis of the anti-cancer susceptibility of Compound A, and the relevance of the presence or absence of p53 and the DNA repair-related genes (ATR, XLF, XRCC4, and LIG4) of Compound A, in which Compound A is a simultaneous inhibitor against PARP/Tankyrase in the human colon cancer cell line HCT116.

(2) FIG. 2 illustrates the results of apoptosis analysis for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, according to a p53 genotype and an LIG4 genotype in various human colon cancer cell lines (RKO, LoVo and SW620).

(3) FIG. 3 illustrates the results of analysis of DNA damage for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in the human colon cancer cell line HCT116 in which the p53 genotype and the LIG4 genotype are normal type, through immunochemical methods.

(4) FIG. 4 illustrates the results of analysis of DNA damage for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in the human colon cancer cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, through immunochemical methods.

(5) FIGS. 5A and 5B illustrate DNA damage and apoptosis for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in human colon cancer cell lines HCT116 and RKO through Western blot.

(6) FIG. 6 illustrates the results of apoptosis analysis for the treatment of Compound A, which is a simultaneous inhibitor against PARP/Tankyrase, after overexpressing mutant type LIG4 (G833A or T1704C) in human colon cancer cell line HCT8 (p53 WT/LIG4 WT).

(7) FIG. 7 illustrates the results of apoptosis analysis for the treatment of Compound A, which is a simultaneous inhibitor against PARP/Tankyrase, overexpressing mutant type LIG4 (G833A or T1704C) in human colon cancer cell line SW620 (p53 MT/LIG4 WT).

(8) FIGS. 8A and 8B illustrate the results of analysis of DNA damage and apoptosis for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, after overexpressing mutant type LIG4 (G833A or T1704C) in human colon cancer cell line HCT8 (p53 WT/LIG4 WT).

(9) FIGS. 9A and 9B illustrate the results of apoptosis analysis for the treatments of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, after overexpressing each of mutant type p53 and normal type LIG4 in human colon cancer cell line RKO (p53 WT/LIG4 MT).

(10) FIGS. 10A and 10B illustrate the results of comparative analysis of the apoptosis for the treatments of Compound A and Compound B, which are the simultaneous inhibitors against PARP/Tankyrase, in the human colon cancer cell line (RKO, LoVo) in which the p53 genotype is the normal type and an LIG4 genotype is the mutant type.

(11) FIGS. 11A to 11D illustrate the results of apoptosis analysis for the treatments of Compound A, Compound B, olaparib and NVP-TNKS656, which are the simultaneous inhibitors against PARP/Tankyrase, in the human colon cancer cell lines RKO (p53 WT/LIG4 MT), SW620 (p53 MT/LIG4 WT), HCT8 (p53 WT/LIG4 WT), and KM12C (p53 MT/LIG4 MT).

(12) FIGS. 12A and 12B illustrate the results of apoptosis analysis for the treatment of Compound B and olaparib, which are the simultaneous inhibitors against PARP/Tankyrase, overexpressing each of the mutant type p53 and normal type LIG4 in the human colon cancer cell line RKO (p53 WT/LIG4 MT).

(13) FIGS. 13A and 13B illustrate the results of apoptosis analysis for the treatment of Compound B and olaparib, which are the simultaneous inhibitors against PARP/Tankyrase, overexpressing each of the mutant type p53 and the normal type LIG4 in the human colon cancer cell line LoVo (p53 WT/LIG4 MT).

(14) FIGS. 14A and 14B illustrate DNA damage and apoptosis for the treatment of Compound B and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, overexpressing each of the mutant type p53 and the normal type LIG4 in the human colon cancer cell line LoVo (p53 WT/LIG4 MT) through Western blot.

(15) FIGS. 15A and 15B illustrate the results of tumor inhibition analysis of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in an in vivo model of xenograft model using the human colon cancer cell line RKO (p53 WT/LIG4 MT).

(16) FIGS. 16A and 16B illustrate the results of tumor inhibition analysis for the administration of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in an in vivo model of xenograft model using the human colon cancer cell line SW620 (p53 MT/LIG4 WT).

(17) FIGS. 17A and 17B illustrate the results of tumor inhibition analysis for the administration of Compound A and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in an in vivo model of xenograft model using the human colon cancer cell line KM12C (p53 MT/LIG4 MT)

(18) FIGS. 18A and 18B illustrate the results of tumor inhibition analysis for the administration of Compound B and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in an in vivo model of xenograft model using the human colon cancer cell line RKO (p53 WT/LIG4 MT).

(19) FIGS. 19A and 19B illustrate the results of tumor inhibition analysis for the administration of Compound B, which is a simultaneous inhibitor against PARP/Tankyrase, in an in vivo model of xenograft model using the human colon cancer cell line KM12C (p53 MT/LIG4 MT).

(20) FIG. 20 shows the results of cell shape change analysis for the treatment of Compound B and olaparib, which are simultaneous inhibitors against PARP/Tankyrase, in colon cancer patients derived cells 11-CT-79558D (p53 WT/LIG4 MT), 11-CT-80464B (p53 WT/LIG4 MT) and 11CT-94575 (p53 WT/LIG4 WT), 13CT-78649B (p53 WT/LIG4 WT).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(21) It will be obvious to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.

Modes of the Invention

(22) Hereinafter, the present disclosure will be described in more detail with reference to the examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present disclosure in more detail and that the scope of the present disclosure is not limited by these embodiments in accordance with the gist of the present invention.

(23) The IUPAC name of the novel PARP/Tankyrase simultaneous inhibitor Compound A used in the present disclosure is 8-[(dimethylamino)methyl]-10-ethoxy-1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridin-5(6H)-one, and the IUPAC name of Compound B is 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile.

Example 1: IC50 Analysis of Compound A as a PARP/Tankyrase Simultaneous Inhibitor

(24) The present inventors conducted an in vitro cell base assay to analyze the anticancer activity of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, in 18 human colon cancer cell lines (Korean Cell Line Bank and ATCC purchased), and the cell viability and IC.sub.50 of the cell lines for Compound A, the novel PARP/Tankyrase simultaneous inhibitor, were measured.

(25) As the experimental conditions and methods, a total of 18 human colon cancer cell lines were cultured in RPMI1640 (Roswell Park Memorial Institute 1640; 10% FBS, 1% penicillin/streptomycin), and each cell line was cultured in 96 well plate for 210.sup.3 per well at 37 C. for 24 hours and the novel PARP/Tankyrase simultaneous inhibitor, Compound A, was diluted by a multiple of 2 at a maximum of 100 M and a minimum of 0.390625 M (100 M, 50 M, 25 M, 12.5 M, 6.25 M, 3.125 M, 1.5625 M, 0.78125 M and 0.390625 M). After the treatment, it was cultured at 37 C. for 72 hours. As for the drug treatment group and non-treatment group, the cell viability of each cell line was measured by MTS analysis (Promega, CellTiter 96 AQeous One Solution) and the IC50 of each cell line was measured using the PRISM program.

(26) Experiment Result

(27) As a result of analyzing IC.sub.50 using MTS analysis of each of the 18 human colon cancer cell lines treated with Compound A, a novel PARP/Tankyrase simultaneous inhibitor, the significant IC.sub.50 values of Compound A were shown in the cell lines of 10 species (HCT116, HT29, RKO, LoVo, LS174T, Colo201, Colo205, Colo320HSR, HCT8 and Caco-2).

(28) TABLE-US-00001 TABLE 1 Colon cancer Compound A Colon cancer Compound A cell line IC.sub.50 (uM) cell line IC.sub.50 (uM) Colo320 8.27 HCT8 39.50 Ls174T 12.19 DLD-1 85.57 HCT116 13.18 SW620 96.65 LoVo 18.57 SW480 >100 Caco-2 19.24 SW1417 >100 RKO 23.71 KM12C >100 Colo205 24.45 SW48 >100 Colo201 28.43 HCT-15 >100 HT-29 32.21 Ls1034 >100

Example 2: Anticancer Susceptibility of Compound A According to p53 Genotype

(29) The present inventors analyzed the correlation of 18 human colon cancer cell lines, an anti-cancer effect of Compound A which is a novel PARP/Tankyrase simultaneous inhibitor and the genotype of the p53 gene through an in vitro cell base assay in the same experimental method as Example 1 in 18 human colon cancer cell lines.

(30) Experiment Result

(31) As a result of the comparison with the IC.sub.50 of Compound A, which is a PARP/Tankyrase simultaneous inhibitor, and the p53 genotype commonly found in colon cancer in 18 human colon cancer cell lines, colon cancer cells with the normal p53 genotype were analyzed with low IC.sub.50 values. The relationship between the p53 genotype and Compound A, a novel PARP/Tankyrase simultaneous inhibitor, was shown.

(32) TABLE-US-00002 TABLE 2 p53 Wild-type IC.sub.50 (M) p53 Mutant type IC.sub.50 (M) cell line Compound A cell line Compound A LS174T 12.19 Colo320HSR 8.267 HT29 32.21 RKO 12.3 Caco-2 19.24 Colo205 24.45 HCT116 13.18 Colo201 28.43 DLD-1 85.57 LoVo 18.57 SW620 96.65 HCT-15 >100 HCT8 39.5 LS1034 >100 KM12C >100 SW48 >100 SW1417 >100 SW480 >100

Example 3: Anticancer Susceptibility Genotype Analysis of Compound A

(33) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, depending on the presence or absence of the p53 gene and the presence or absence of the DNA repair gene, the present inventors reduced gene expression by an ATR, XLF, XRCC4 and LIG4 siRNA knockdown method known as a DNA repair gene in the human colon cancer cell line HCT116 in which the p53 normal gene is present, and then treated with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. As the experimental conditions and methods, the HCT116 p53 null human colon cancer cell line in which the p53 gene is normal and the HCT116 and p53 gene are deficient is cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin), and 110.sup.5 per well was cultured at 37 C. for 24 hours in a 60 mm plate. ATR, XLF, XRCC4 and LIG4 (SEQ ID NO: 4; ATR siRNA 5-GAGUUCUCAGAAGUCAACC-3, SEQ ID NO: 5; XLF siRNA 5-CGCUGAUUCGAGAUCGAUUGA-3, SEQ ID NO: 6; XRCC4 siRNA 5-CUGAUCUCUCUGGGUUGGCUU-3, SEQ ID NO: 7; LIG4 siRNA 5-GGGAGUGUCUCAUGUAAUA-3) were infused into cells by means of Lipofectamin 2000 (Invitrogen), and knocked down at 37 C. for 48 hours. The cells were treated with 25 M and 50 M of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor. After 48 hours of culturing, the number of cells was measured by trypan blue staining and the degree of apoptosis was confirmed.

(34) Experiment Result

(35) DNA repair-related genes were knocked down according to the presence or absence of p53 gene in HCT116 and p53-deficient HCT116 (HCT116 p53 null), which are colon cancer cell lines of normal genotype p53. As a result of analyzing a DNA repair gene which shows the susceptibility only in Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, in case where the p53 gene is normal and the LIG gene is deficient, the apoptosis is significantly increased, thus showing a selective result in Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 1).

Example 4: Analysis of LIG4 Genotype in Colon Cancer Cells

(36) In order to confirm an LIG4 genotype in the colon cancer cell line, the present inventors analyzed the mutation/deficiency of an LIG4 genotype in 20 colon cancer cell lines through RT-PCR and sequencing. The conditions and methods of the experiment were as follows: A total of 20 colon cancer cell lines were subjected to total RNA extraction with Trizol RNA extraction method using a homogenizer, and 500 ng of total RNA was re-synthesized into cDNA and PCR was performed using LIG4 primer (SEQ ID NO: 8; LIG4 primer Exon2-1 forward primer 5-GCTAGCTGCTATTGCAGATATTGAGC-3, SEQ ID NO: 9; LIG4 primer Exon2-1 reverse primer 5-AGAACCTTCAGTAGGAGAAGCACCAA-3, SEQ ID NO: 10; LIG4 primer Exon2-2 forward primer Exon2-2 forward primer 5-CCTGGTGAGAAGCCATCTGT-3, SEQ ID NO: 11; LIG4 primer Exon2-2 reverse primer 5-GCCTTCCCCCTAAGTTGTTC-3). After electrophoresis on 1% agarose gel, the mutant analysis was confirmed by sanger sequencing of the PCR product in which the expression of LIG4 was confirmed through Et-Br staining.

(37) Experiment Result

(38) In order to confirm whether there is any mutation of LIG4 genotype in 20 human colon cancer cell lines, the G833 and T1704 sites, which are known as malfunction mutation sites of the LIG4 gene, were analyzed by the singer sequencing method. As a result, it has been confirmed that there is a mutation of LIG4 genotype in 8 colon cancer cell lines among 20 thereof.

(39) TABLE-US-00003 TABLE 3 Colon cancer cells SW460 SW620 HCT116 HT29 RKO DLO1 SW1417 Colo320HSR LIG4 mutation 833G>A WT WT WT WT WT WT WT WT (DNA ligase4) 1704T>C WT WT WT WT Mut WT WT Mut Colon cancer cells Ls174T HCT8 L91034 Colo201 LOVO SNUC2A KM12C HCT15 LIG4 mutation 833G>A Mut WT WT WT Mut WT WT Mut (DNA ligase4) 1704T>C WT WT WT WT WT WT Mut Mut Colon cancer cells CaCO2 KM12CL4 Colo205 SW48 LIG4 mutation 833G>A Mut WT WT WT (DNA ligase4) 1704T>C WT Mut WT WT

Example 5: Analysis of Apoptosis According to p53 and LIG4 Genotypes

(40) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor according to a p53 genotype and LIG4 genotype, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and a competitive drug PARP inhibitor, olaparib, in RKO, LoVo human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a mutant type and an SW620 human colon cancer cell line in which the p53 gene is a mutant type and the LIG4 gene is a normal type. The number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The conditions and methods of the experiment were as follows: RKO, LoVo human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a mutant type and the SW620 human colon cancer cell line in which the p53 gene is a mutant type and the LIG4 gene is a normal type were cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with each of 25 M and 50 M and were cultured for 48 hours. The number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis.

(41) Experiment Result

(42) As a result of observing the degree of apoptosis by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in a human colon cancer cell line whose p53 and LIG4 genotypes are difference, the apoptosis was induced by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, in the RKO, LoVo human colon cancer cell line in which the p53 gene is a normal type and an LIG4 gene is a mutant type, and no apoptosis effect was shown in olaparib, which is a competitive drug PARP inhibitor. In the SW620 human colon cancer cell line in which the p53 gene is a mutant type and an LIG4 gene is a normal type, it was observed that both Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib showed low apoptosis effect, thus showing a selective result in Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor in which the p53 genotype is normal and an LIG4 genotype is a mutant type (FIG. 2).

Example 6: DNA Damage Analysis of p53 Genotype Normal Type Cells

(43) In order to analyze the degree of DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor when the p53 gene is a normal type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and the competitive drug PARP inhibitor, olaparib, in the HCT116 human colon cancer cell line in which the p53 gene is a normal type, and then the DNA damage was confirmed by immunochemical method as the expression level of r-H2AX. The conditions and methods of the experiment were as follows: HCT116 human colon cancer cell line in which the p53 gene is a normal type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with 50 M each and were cultured for 48 hours. After being fixed with 4% paraformaldehyde for 15 minutes at room temperature and being washed with TBS-T buffer three times for 10 minutes, they were permeabilized with 0.5% Triton X-100 for 10 minutes. After being washed three times for 10 minutes with TBS-T buffer, they were blocked with 5% BSA for 1 hour at room temperature. The r-H2AX antibody was diluted at a ratio of 1:100 in 1% BSA, reacted overnight at 4 C., washed three times for 10 minutes with TBS-T buffer, and the secondary antibody (alexa Fluor488 fitc) was diluted at a ratio of 1:100 in 1% BSA, reacted at room temperature for 2 hours, and then washed with TBS-T A buffer three times for 10 minutes, and stained with DAPI (4,6-diamidino-2-phenylindole) to confirm the expression level of r-H2AX under a fluorescence microscope.

(44) Experiment Result

(45) As a result of observing the DNA damage with the expression level of r-H2AX by imuunocytochemistry method by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in a human colon cancer cell lineHCT116 in which the p53 gene is a normal type, a group treated with Compound A, the novel PARP/Tankyrase simultaneous inhibitor, showed a higher expression of r-H2AX than the group treated with olaparib, thus inducing DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 3).

Example 7: DNA Damage Analysis of p53 Genotype and LIG4 Genotype Mutant Cells

(46) In order to analyze the degree of DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, when the p53 gene is a normal type and the LIG4 gene is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and the competitive drug PARP inhibitor, olaparib, in the RKO human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a mutant type, and the DNA damage was confirmed by the immunochemical method as the expression level of r-H2AX. The conditions and methods of the experiment were as follows: RKO human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a mutant type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with 50 M each and were cultured for 48 hours. After being fixed with 4% paraformaldehyde for 15 minutes at room temperature and being washed with TBS-T buffer ten times for 5 minutes, they were permeabilized with 0.5% Triton X-100 for 10 minutes. After being washed three times for 10 minutes with TBS-T buffer, they were blocked with 5% BSA for 1 hour at room temperature. The r-H2AX antibody was diluted at a ratio of 1:100 in 1% BSA, reacted overnight at 4 C., washed three times for 10 minutes with TBS-T buffer, and the secondary antibody (Alexa Fluor488 fitc) was diluted at a ratio of 1:100 in 1% BSA, reacted at room temperature for 2 hours, and then washed with TBS-T A buffer three times for 10 minutes, and stained with DAPI to confirm the expression level of r-H2AX under a fluorescence microscope.

(47) Experiment Result

(48) As a result of observing the DNA damage with the expression level of r-H2AX by imuunocytochemistry method by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in the RKO human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a mutant type, the expression of r-H2AX was increased only in the group treated with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, indicating that it induces DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 4).

Example 8: DNA Damage and Apoptosis Analysis of p53 Genotype Normal Type and LIG4 Genotype Mutant Type Cells

(49) In order to analyze the degree of DNA damage and the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, when the p53 gene is a normal type and the LIG4 gene is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and the competitive drug PARP inhibitor; olaparib, in the HCT116 human colon cancer cell line in which the p53 gene and an LIG4 gene are normal types and in the RKO human colon cancer cell line in which the p53 gene is a normal type and an LIG4 gene is a mutant type, and then confirmed the DNA damage with the expression level of r-H2Ax by western blotting method and confirmed the degree of apoptosis with the expression level of cleaved caspase 3. The conditions and method of the experiment were as follows: The HCT116 human colon cancer cell line in which the p53 gene and an LIG4 gene are normal types and the RKO human colon cancer cell line in which the p53 gene is a normal type and an LIG4 gene is a mutant type were cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with 50 M each and were cultured for 48 hours. Then, the cells were obtained using a centrifuge. Each cell thus obtained was dissolved using an RIPA buffer, and the proteins were extracted using a high-speed centrifuge. 30 g protein per cell was electrophoresed by western blot method to separate proteins and transferred to PDVF membrane. The r-H2AX, truncated caspase 3, and b-actin antibody were diluted with each 1:2000 in 5% skim milk. Then, the cells were reacted at 4 C. for 12 hours and then washed three times for 15 minutes with TBS-T buffer. The secondary antibody was diluted with each 1:2000 ratio in 5% skim milk and reacted at room temperature for 2 hours. It was washed with TBS-T buffer three times for 15 minutes to induce luminescence of PDVF membrane using ECL (Enhanced Chemiluminescence) buffer solution to develop protein expression on each anti-body using an X-ray film.

(50) Experiment Result

(51) As a result of observing the degree of DNA damage and apoptosis with the expression levels of r-H2AX and truncated caspase 3 by western blotting by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, in the human colon cancer cell line HCT116 in which the p53 genotype is a normal type and the human colon cancer cell line RKO in which an LIG4 genotype is a mutant type, the group treated with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, showed an increased expression of r-H2AX than the group treated with olaparib, and showed an increased expression amount of the truncated caspase 3, thus inducing DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and apoptosis (FIG. 5).

Example 9: Analysis of Apoptosis in the Case where an LIG4 Genotype is a Mutant Type Cell in a Cell Line where the p53 Genotype is a Normal Type

(52) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and the competitive drug PARP inhibitor, olaparib, while overexpressing an LIG4 mutant type in the HCT8 human colon cancer cell line in which the p53 gene is a normal type and an LIG4 genotype is a normal type. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The conditions and methods of the above experiment were as follows: HCT8 human colon cancer cell line in which the p53 gene is a normal type and the LIG4 gene is a normal type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. G833A or T1704C plasmid DNA in which an LIG4 genotype is a mutant type was infused into cells by means of Lipofectamin 2000 (Invitrogen), and then overexpressed at 37 C. for 48 hours. Then, the cells were treated with each of 25 M and 50 M of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor and olaparib, a competitive drug PARP inhibitor. After 48 hours of culturing, the number of cells was measured by trypan blue staining and the degree of apoptosis was confirmed.

(53) Experiment Result

(54) As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor while expressing G833A or T1704C in which an LIG4 genotype is a mutant type in the human colon cancer cell line HCT8 in which the p53 genotype and an LIG4 genotype is a normal type, as compared to a control group (empty) into which an LIG4 mutant gene is not inserted, in case where an LIG4 mutant G833A or T1704C is overexpressed, it showed that the apoptosis was increased by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 6).

Example 10: Analysis of Apoptosis in the Case of LIG4 Genotype Mutant Cells in a Cell Line in which the p53 Genotype is a Mutant Type

(55) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, when the p53 genotype is a mutant type and an LIG4 genotype is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound A, and the competitive drug PARP inhibitor, olaparib, while overexpressing an LIG4 mutant type in the SW620 human colon cancer cell line in which the p53 gene is a mutant type and an LIG4 genotype is a normal type. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The conditions and methods of the above experiment were as follows: SW620 human colon cancer cell line in which the p53 gene is a mutant type and the LIG4 gene is a normal type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. G833A or T1704C plasmid DNA in which an LIG4 genotype is a mutant type was infused into cells by means of Lipofectamin 2000 (Invitrogen), and then overexpressed at 37 C. for 48 hours. Then, the cells were treated with each of 25 M and 50 M of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor and olaparib, a competitive drug PARP inhibitor. After 48 hours of culturing, the number of cells was measured by trypan blue staining and the degree of apoptosis was confirmed.

(56) Experiment Result

(57) As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor while expressing G833A or T1704C in which an LIG4 genotype is a mutant type in the human colon cancer cell line SW620 in which the p53 genotype is a mutant type and an LIG4 genotype is a normal type, as compared to a control group (empty) into which an LIG4 mutant gene is not inserted, in case where an LIG4 mutant G833A or T1704C is overexpressed, no change in the apoptosis was shown by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor. As illustrated in FIG. 5, only in case where the p53 genotype is a normal type, the apoptosis was induced by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor for an LIG4 mutant (FIG. 7).

Example 11: Analysis of DNA Damage and Apoptosis in LIG4 Genotype Mutant Cells in the Cell Line in which the p53 Genotype is a Normal Type

(58) The present inventors conducted an experiment in the same manner as Example 9 to analyze the degree of apoptosis and DNA damage of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, when the p53 genotype is a mutant type and an LIG4 genotype is a mutant type. The number of cells was measured by trypan blue staining method and the degree of apoptosis was confirmed. The cells were obtained using a centrifuge. Each cell thus obtained was dissolved using an RIPA buffer, and the proteins were extracted using a high-speed centrifuge. 30 g protein per cell was electrophoresed by western blot method to separate proteins and transferred to PDVF membrane. The r-H2AX, truncated caspase 3, and b-actin antibody were diluted with each 1:2000 ratio in 5% skim milk. Then, the cells were reacted at 4 C. for 12 hours and then washed three times for 15 minutes with TBS-T buffer. The secondary antibody was diluted with each 1:2000 ratio in 5% skim milk and reacted at room temperature for 2 hours. It was washed with TBS-T buffer three times for 15 minutes to induce luminescence of PDVF membrane using ECL buffer solution to develop protein expression on each anti-body using an X-ray film.

(59) Experiment Result

(60) As a result of observing the degree of DNA damage and the degree of apoptosis by r-H2AX and the expression level of truncated caspase 3 by western blot and observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor and olaparib, which is a competitive drug PARP inhibitor, while overexpressing G833A or T1704C in which an LIG4 genotype is a mutant type in the human colon cancer cell line HCT8 in which the p53 genotype and an LIG4 genotype is a normal type, the group treated with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, showed an increased apoptosis by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor in case of overexpressing the LIG4 mutant G833A or T1704C as compared to a control group (empty) into which an LIG4 mutant gene is not inserted, rather than olaparib, which is a competitive drug (FIG. 8A). The expression of r-H2AX and the expression amount of truncated caspase 3 were also increased, indicating that only when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the DNA damage and apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, were induced (FIG. 8B)

Example 12: Analysis of Apoptosis by p53 Mutant Type and LIG4 Normal Type Expression in a Cell Line in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(61) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor according to the expression of mutant type p53 and normal type LIG4 when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the RKO human colon cancer cell line in which the p53 gene is a normal type and an LIG4 genotype is a mutant type was cultured in the same manner as in Example 7. The p53 mutant type plasmid DNA and the plasmid DNA in which an LIG4 genotype is a normal type were infused into the cells for 48 hours for overexpression at 37 C., and then Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparip, which is a competitive drug PARP inhibitor, were treated with 25 M and 50 M, respectively, for 48 hours. The number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis.

(62) Experiment Result

(63) As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing the p53 mutant type gene in the human colon cancer cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where the p53 mutant type gene was overexpressed as compared to a control group (empty) into which the p53 mutant type gene is not inserted, it was observed that the apoptosis was reduced by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 9A). As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing an LIG4 normal type gene in the human colon cancer cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where an LIG4 normal type gene was overexpressed as compared to a control group (empty) into which an LIG4 normal type gene is not inserted, it was observed that the apoptosis was reduced by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 9B). When the p53 is a normal type and an LIG4 is a mutant type, the apoptosis of Compound A is the highest (FIG. 9).

Example 13: IC.SUB.50 .Analysis of Compound B which is a PARP/Tankyrase Simultaneous Inhibitor According to p53 and LIG4 Genotypes

(64) In order to analyze the anticancer activity of Compounds A and B, which are PARP/Tankyrase simultaneous inhibitors, in four types of human colon cancer cell lines of RKO (p53 WT/LIG4 MT), HCT8 (p53 WT, LIG4 WT), SW620 (p53 MT, LIG4 WT), and KM12C (p53 MT, LIG4 MT) with different p53 and LIG4 genotypes, the present inventors measured the cell viability and IC.sub.50 of cell lines for Compounds A and B, which are PARP/Tankyrase simultaneous inhibitors, through in vitro cell base assay. The conditions and methods of the experiment were as follows: Four types of human colon cancer cell lines were cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin), and each cell line was cultured in 96 well plate for 210.sup.3 per well at 37 C. for 24 hours, and the novel PARP/Tankyrase simultaneous inhibitor, Compound A, was diluted by a multiple of 2 at a maximum of 100 M and a minimum of 0.390625 M (100 M, 50 M, 25 M, 12.5 M, 6.25 M, 3.125 M, 1.5625 M, 0.78125 M and 0.390625 M). After the treatment, it was cultured at 37 C. for 72 hours. As for the drug treatment group and non-treatment group, the cell viability of each cell line was measured by MTS analysis (Promega, CellTiter 96 AQeous One Solution) and the IC50 of each cell line was measured using the PRISM program.

(65) Experiment Result

(66) As a result of analyzing IC.sub.50 using MTS analysis of each of the 4 human colon cancer cell lines treated with Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, the lowest IC.sub.50 values of Compounds A and B was shown in the RKO in which p53 is a normal type and an LIG4 is a mutant type among 4 types of cell lines, indicating that Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, show the most excellent anticancer effect in case where p53 is a normal type and LIG4 is a mutant type (Table 4).

(67) TABLE-US-00004 TABLE 4 Colon cancer cell (IC.sub.50, uM) RKO HCT8 SW620 KM12C Compound (p53wt/ (p53wt/ (p53mt/ (p53mt/ Name LIG4mt) LIG4wt) LIG4wt) LIG4mt) Compound A 23.71 39.5 96.65 >100 Compound B 12.46 62.52 >100 >100

Example 14: Analysis of Efficacy of Compound A and Compound B According to p53 and LIG4 Genotypes

(68) In order to analyze the degree of apoptosis of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, according to the p53 genotype and an LIG4 genotype, the present inventors treated Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, in the RKO and LoVo human colon cancer cell lines in which the p53 gene is a normal type and an LIG4 gene is a mutant type. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The RKO and LoVo human colon cancer cell lines were cultured under the same conditions as in Example 5. Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, were treated with 25 M and 50 M, respectively, for 48 hours. After 48 hours of culturing, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis.

(69) Experiment Result

(70) As a result of observing the degree of apoptosis by treating each of Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, in the human colon cancer cell lines RKO and LoVo in which the p53 gene is a normal type and an LIG4 gene is a mutant type, in the RKO and LoVo cell lines in which the p53 gene is a normal type and an LIG4 gene is a mutant type, it is shown that the apoptosis effect is high by virtue of Compound B as compared to Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor. In case where the p53 genotype is normal and an LIG4 genotype is a mutant type, Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, showed a selective result as compared to Compound A.

Example 15: Analysis of Apoptosis of Compound A and Compound B in Cell Lines Different in p53 Genotype and LIG4 Genotype

(71) In order to analyze the degree of apoptosis of Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, in a cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, a cell line SW620 in which the p53 genotype is a mutant type and an LIG4 genotype is a normal type, a cell line HCT8 in which the p53 genotype and an LIG4 genotype are normal, and a cell line KM12C in which the p53 genotype and an LIG4 genotype are mutant, the human colon cancer cell lines of RKO, HCT, SW620 and KM12C with different p53 genotype and LIG4 genotype were cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin) and cultured in a 60 mm plate at 110.sup.5 per well for 24 hours at 37 C. Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, and olaparib, which is a competitive drug PARP inhibitor, and NVP-TNKS656, which is a Tankyrase inhibitor, were treated with each of 25 M and 50 M. After 48 hours of culturing, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis.

(72) Experiment Result

(73) As a result of analyzing the degree of apoptosis by trypan blue straining exclusion assay by treating Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, olaparib, which is a competitive drug PARP inhibitor, and NVP-TNKS656, which is a Tankyrase inhibitor in the human colon cancer cell lines RKO, SW620, HCT8 and KM12C with different p53 genotype and LIG4 genotype, it was possible to observe the apoptosis of Compounds A and B, which are novel PARP/Tankyrase simultaneous inhibitors, in the cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type. Also, it was observed that there was no change in apoptosis by olaparib, which is a competitive drug PARP inhibitor, and NVP-TNKS656, which is a Tankyrase inhibitor (FIG. 11A). No apoptosis occurred by Compounds A and B, olaparib, which is a PARP inhibitor, and NVP-TNKS656, which is a Tankyrase inhibitor in the cell line SW620 in which p53 is a mutant type and LIG4 is a normal type, the cell line HCT8 in which p53 and LIG4 genotypes are normal types, and the cell line KM12C in which p53 and LIG4 genotypes are mutant types, indicating that the apoptosis was shown by Compounds A and B only in case where p53 is a normal type and LIG4 is a mutant type (FIGS. 11A to 11D).

Example 16: Analysis of the Apoptosis of Compound B According to p53 Mutant Type and LIG4 Normal Type Overexpression in the Cell Line RKO in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(74) As in Example 12, in order to analyze the degree of apoptosis of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, according to the expression of a mutant type p53 and a normal type LIG4 when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound B, and the competitive drug PARP inhibitor, olaparib, while overexpressing the p53 mutant type and the LIG4 normal type in the RKO human colon cancer cell line in which the p53 gene is a normal type and an LIG4 genotype is a mutant type. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The conditions and methods of the above experiment were as follows: RKO human colon cancer cell line in which the p53 gene is a normal type and an LIG4 gene is a mutant type was cultured under the same condition as in Example 12. p53 mutant type plasmid DNA and plasmid DNA in which an LIG4 genotype is a normal type were infused into cells by means of Lipofectamin 2000 (Invitrogen), and then overexpressed at 37 C. for 48 hours. Then, Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with each of 25 M and 50 M. After 48 hours of culturing, the number of cells was measured by trypan blue staining assay and the degree of apoptosis was confirmed.

(75) Experiment Result

(76) As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing the p53 mutant type gene in the human colon cancer cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where the p53 mutant type gene was overexpressed as compared to a control group (empty) into which the p53 mutant type gene is not inserted, it was observed that the apoptosis was reduced by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 12A). As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing an LIG4 normal type gene in the human colon cancer cell line RKO in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where an LIG4 normal type gene was overexpressed as compared to a control group (empty) into which an LIG4 normal type gene is not inserted, it was observed that the apoptosis was reduced by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor. When the p53 is a normal type and an LIG4 is a mutant type, the apoptosis of Compound Bis the highest (FIGS. 12A and 12B).

Example 17: Analysis of the Apoptosis of Compound B According to p53 Mutant Type and LIG4 Normal Type Overexpression in the Cell Line LoVo in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(77) As in Example 12, in order to analyze the degree of apoptosis of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, according to the expression of a mutant type p53 and a normal type LIG4 when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors treated a novel PARP/Tankyrase simultaneous inhibitor, Compound B, and the competitive drug PARP inhibitor, olaparib, while overexpressing the p53 mutant type and the LIG4 normal type in the LoVo human colon cancer cell line in which the p53 gene is a normal type and an LIG4 genotype is a mutant type. Then, the number of cells was measured by trypan blue staining assay to confirm the degree of apoptosis. The conditions and methods of the above experiment were the same as the methods of RKO cell culturing and transfection methods of Example 12. Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were treated with each of 25 M and 50 M. After 48 hours of culturing, the number of cells was measured by trypan blue staining assay and the degree of apoptosis was confirmed.

(78) Experiment Result

(79) As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing the p53 mutant type gene in the human colon cancer cell line LoVo in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where the p53 mutant type gene was overexpressed as compared to a control group (empty) into which the p53 mutant type gene is not inserted, it was observed that the apoptosis was reduced by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 13A). As a result of observing the degree of apoptosis by trypan blue straining exclusion assay by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing an LIG4 normal type gene in the human colon cancer cell line LoVo in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where an LIG4 normal type gene was overexpressed as compared to a control group (empty) into which an LIG4 normal type gene is not inserted, it was observed that the apoptosis was reduced by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 13B). When the p53 is a normal type and an LIG4 is a mutant type, the apoptosis of Compound B is the highest (FIGS. 13A and 13B).

Example 18: DNA Damage and Apoptosis Analysis of Compound B According to p53 Mutant Type and LIG4 Normal Type Overexpression in the Cell Line LoVo in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(80) In order to analyze the degree of DNA damage and the degree of apoptosis of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, according to the expression of a mutant type p53 and a normal type LIG4 when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors cultured and treated the LoVo human colon cancer cell line in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type under the same condition as in Example 17. DNA damage was confirmed by the Western blot method at the expression level of r-H2AX, and the degree of apoptosis was confirmed by the same method as Example 11 at the expression level of cleaved caspase 3.

(81) Experiment Result

(82) As a result of observing the degree of DNA damage and the degree of apoptosis by western blot by means of the expression amount of r-H2AX and cleaved caspase 3 by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing the p53 mutant type gene in the human colon cancer cell line LoVo in which the p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where the p53 mutant type gene was overexpressed as compared to a control group (empty) into which the p53 mutant type gene is not inserted, it was observed that the expression of r-H2AX and the expression amount of cleaved caspase 3 were reduced by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 14A). As a result of observing the degree of DNA damage and the degree of apoptosis by western blot by means of the expression amount of r-H2AX and cleaved caspase 3 by treating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, while overexpressing the p53 mutant type gene in the human colon cancer cell line LoVo in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type, in case where an LIG4 normal type gene was overexpressed as compared to a control group (empty) into which an LIG4 normal type gene is not inserted, it was observed that the expression of r-H2AX and the expression amount of cleaved caspase 3 were reduced by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor (FIG. 14B). Only when a p53 is a normal type and an LIG4 is a mutant type, the DNA damage and apoptosis of Compound B were induced (FIG. 14).

Example 19: Tumor Inhibition Effect on Compound A and Olaparib in a Xenotransplantation Animal Model Using a Cell Line RKO in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(83) In order to analyze the tumor inhibition effect on an in vivo animal model for Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and an in vivo animal model for olaparib, when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors transplanted the RKO human colon cancer cell line in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type into a 6-week-old nude mouse (BALB/c-nude, purchased from the central experimental animal) and administered with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor. Then, the size of tumor was measured to confirm the degree of tumor inhibition effect. The conditions and methods of the experiment were as follows: RKO human colon cancer cell line in which a p53 gene is a normal type and an LIG4 gene is a mutant type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin). When the size of the tumor reached 100 mm.sup.3 after being transplanted into a nude mouse per 110.sup.7, Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were orally administered at 25 mpk and 50 mpk daily for a total of 27 days. The size of a tumor was measured every three days. After the completion of drug administration, the tumors were harvested and weighed.

(84) Experiment Result

(85) As a result of observing the degree of a tumor inhibition effect by administrating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in the human colon cancer cell line RKO in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type, the tumor inhibition effect of Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, the size of the tumor was reduced compared with the group administered with olaparib, which is a competitive drug PARP inhibitor, and the weight of a tumor was reduced, indicating that the tumor inhibition effect for Compound A was produced in case where p53 is a normal type and LIG4 is a mutant type (FIGS. 15A and 15B).

Example 20: Tumor Inhibition Effect Analysis on Compound A and Olaparib in a Xenotransplantation Animal Model Using a Cell Line SW620 in which the p53 Genotype is a Mutant Type and an LIG4 Genotype is a Normal Type

(86) In order to analyze the tumor inhibition effect on Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, in an in vivo animal model when the p53 genotype is a mutant type and an LIG4 genotype is a normal type, the present inventors transplanted the SW620 human colon cancer cell line in which a p53 genotype is a mutant type and an LIG4 genotype is a normal type into a 6-week-old nude mouse (BALB/c-nude, purchased from the central experimental animal) and administered with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor. Then, the size of tumor was measured to confirm the degree of tumor inhibition effect. The conditions and methods of the experiment were as follows: SW620 human colon cancer cell line in which a p53 gene is a mutant type and an LIG4 gene is a normal type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin). When the size of the tumor reached 100 mm.sup.3 after being transplanted into a nude mouse per 110.sup.7, Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were orally administered at 25 mpk and 50 mpk daily for a total of 27 days. The size of a tumor was measured every three days. After the completion of drug administration, the tumors were harvested and weighed.

(87) Experiment Result

(88) As a result of observing the degree of a tumor inhibition effect by administrating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in the human colon cancer cell line SW620 in which a p53 genotype is a mutant type and an LIG4 genotype is a normal type, there was no change both in tumor size and weight by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, indicating that no tumor inhibition effect for Compound A and olaparib was produced in case where p53 is a normal type and LIG4 is a mutant type (FIGS. 16A and 16B).

Example 21: Analysis of Tumor Inhibition Effect on Compound A in a Xenotransplantation Animal Model Using a Cell Line KM12C in which a p53 Genotype and an LIG4 Genotype are Mutant Types

(89) In order to analyze the tumor inhibition effect on Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, in an in vivo animal model when a p53 genotype and an LIG4 genotype are mutant types, the present inventors transplanted the KM12C human colon cancer cell line in which a p53 genotype and an LIG4 genotype are mutant types into a 6-week-old nude mouse (BALB/c-nude, purchased from the central experimental animal) and administered with Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor. Then, the size of tumor was measured to confirm the degree of tumor inhibition effect. The conditions and methods of the experiment were as follows: KM12C human colon cancer cell line in which a p53 genotype and an LIG4 genotype are mutant types was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin). When the size of the tumor reached 100 mm.sup.3 after being transplanted into a nude mouse per 110.sup.7, Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were orally administered at 25 mpk and 50 mpk daily for a total of 18 days. The size of a tumor was measured every three days. After the completion of drug administration, the tumors were harvested and weighed.

(90) Experiment Result

(91) As a result of observing the degree of a tumor inhibition effect by administrating Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in the human colon cancer cell line KM12C in which a p53 genotype and an LIG4 genotype are mutant types, there was no change both in tumor size and weight by Compound A, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, indicating that no tumor inhibition effect for Compound A and olaparib was produced in case where both p53 and LIG4 are mutant types (FIGS. 17A and 17B).

Example 22: Analysis of Tumor Inhibition Effect on Compound B and Olaparib in a Xenotransplantation Animal Model Using a Cell Line RKO in which the p53 Genotype is a Normal Type and an LIG4 Genotype is a Mutant Type

(92) In order to analyze the tumor inhibition effect on an in vivo animal model for Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, when the p53 genotype is a normal type and an LIG4 genotype is a mutant type, the present inventors transplanted the RKO human colon cancer cell line in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type into a 6-week-old nude mouse (BALB/c-nude, purchased from the central experimental animal) and administered with Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor. Then, the size of tumor was measured to confirm the degree of tumor inhibition effect. The conditions and methods of the experiment were as follows: RKO human colon cancer cell line in which a p53 gene is a normal type and an LIG4 gene is a mutant type was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin). When the size of the tumor reached 100 mm.sup.3 after being transplanted into a nude mouse per 110.sup.7, Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor, were orally administered at 25 mpk and 50 mpk daily for a total of 27 days. The size of a tumor was measured every three days. After the completion of drug administration, the tumors were harvested and weighed.

(93) Experiment Result

(94) As a result of observing the degree of a tumor inhibition effect by administrating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor in the human colon cancer cell line RKO in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type, the tumor inhibition effect of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, the size of the tumor was reduced compared with the group administered with olaparib, which is a competitive drug PARP inhibitor, and the weight of a tumor was reduced, indicating that the tumor inhibition effect for Compound B was produced in case where p53 is a normal type and LIG4 is a mutant type (FIGS. 18A and 18B).

Example 23: Analysis of Tumor Inhibition Effect on Compound B in a Xenotransplantation Animal Model Using a Cell Line KM12C in which a p53 Genotype and an LIG4 Genotype are Mutant Types

(95) In order to analyze the tumor inhibition effect on Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, in an in vivo animal model when a p53 genotype and an LIG4 genotype are mutant types, the present inventors transplanted the KM12C human colon cancer cell line in which a p53 genotype and an LIG4 genotype are mutant types into a 6-week-old nude mouse (BALB/c-nude, purchased from the central experimental animal) and administered with Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor. Then, the size of tumor was measured to confirm the degree of tumor inhibition effect. The conditions and methods of the experiment were as follows: KM12C human colon cancer cell line in which a p53 genotype and an LIG4 genotype are mutant types was cultured in RPMI1640 (10% FBS, 1% penicillin/streptomycin). When the size of the tumor reached 100 mm.sup.3 after being transplanted into a nude mouse per 110.sup.7, Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, was orally administered at 25 mpk and 50 mpk daily for a total of 15 days. The size of a tumor was measured every three days. After the completion of drug administration, the tumors were harvested and weighed.

(96) Experiment Result

(97) As a result of observing the degree of a tumor inhibition effect by administrating Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, in the human colon cancer cell line KM12C in which in which a p53 genotype and an LIG4 genotype are mutant types, there was no change both in tumor size and weight by Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, indicating that no tumor inhibition effect for Compound B was produced in case where both p53 and LIG4 are mutant types (FIGS. 19A and 19B).

Example 24: Analysis of LIG4 Genotype in Colon Cancer Patient-Derived Cells

(98) In order to confirm an LIG4 genotype in the colon cancer patient-derived cells, the present inventors analyzed the mutation/deficiency of an LIG4 genotype in 36 colon cancer patient-derived cell lines through RT-PCR and sequencing. The conditions and methods of the experiment were as follows: A total of 36 colon cancer patient-derived cell lines were subjected to total RNA extraction with Trizol RNA extraction method using a homogenizer, and 500 ng of total RNA was re-synthesized into cDNA and PCR was performed using LIG4 primer (SEQ ID NO: 12; LIG4 primer Exon1-1 forward primer 5-TTGCTTTACTAGTTAAACGAGAAGATTCA-3, SEQ ID NO: 13; LIG4 primer Exon1-1 reverse primer 5-TTCGTTCTAAAGTTGAACACAAATCTG-3, SEQ ID NO: 8; LIG4 primer Exon2-1 forward primer 5-GCTAGCTGCTATTGCAGATATTGAGC-3, SEQ ID NO: 9; LIG4 primer Exon2-1 reverse primer 5-AGAACCTTCAGTAGGAGAAGCACCAA-3, SEQ ID. NO.: 10; LIG4 primer Exon2-2 forward primer 5-CCTGGTGAGAAGCCATCTGT-3, SEQ ID. NO.: 11; LIG4 primer Exon2-2 reverse primer 5-GCCTTCCCCCTAAGTTGTTC-3). After electrophoresis on 1% agarose gel; the mutant analysis was confirmed by Sanger sequencing of the PCR product in which the expression of LIG4 was confirmed through Et-Br staining.

(99) Experiment Result

(100) As a result of analyzing whether there is any mutation in the C8, C27, G833, and T1704 sites of LIG4 in 36 colon cancer patient-derived cell lines, mutations in LIG4 were analyzed in 14 colon cancer patient-derived cell lines (Table 5).

(101) TABLE-US-00005 TABLE 5 Human colorectal primary cancer cell LIG4 (DNA ligase IV) mutation No. C8T C26T G833A T1704C 1 WT WT WT WT 2 WT MT WT WT 3 WT MT WT WT 4 WT MT WT WT 5 WT WT WT WT 6 WT MT WT WT 7 WT MT WT WT 8 WT WT WT WT 9 WT WT WT WT 10 WT WT WT WT 11 WT WT WT WT 12 WT MT WT WT 13 WT WT WT WT 14 WT WT WT WT 15 WT WT WT WT 16 WT MT WT WT 17 WT WT WT WT 18 WT WT WT WT 19 WT WT WT WT 20 WT WT WT WT 21 WT WT WT WT 22 WT WT WT WT 23 WT MT WT WT 24 WT MT WT WT 25 WT WT WT WT 26 WT WT WT WT 27 WT WT WT WT 28 WT WT WT WT 29 WT MT WT WT 30 WT MT WT WT 31 MT WT WT WT 32 WT WT WT WT 33 WT MT WT WT 34 WT WT WT WT 35 WT MT WT WT 36 WT WT WT WT

Example 25: Analysis of LIG4 Genotypes in Colon Cancer Patient Tissues

(102) In order to confirm an LIG4 genotype in the colon cancer patient tissues, the present inventors analyzed the mutation/deficiency of an LIG4 genotype in 39 colon cancer patient-derived tissues through RT-PCR and sequencing. The conditions and methods of the experiment were as follows: A total of 39 colon cancer patient-derived tissues were subjected to total RNA extraction with Trizol RNA extraction method using a homogenizer, and 500 ng of total RNA was re-synthesized into cDNA and PCR was performed using LIG4 primer (SEQ ID NO: 12; LIG4 primer Exon1-1 forward primer 5-TTGCTTTACTAGTTAAACGAGAAGATTCA-3, SEQ ID NO: 13; LIG4 primer Exon1-1 reverse primer 5-TTCGTTCTAAAGTTGAACACAAATCTG-3, SEQ ID NO: 8; LIG4 primer Exon2-1 forward primer 5-GCTAGCTGCTATTGCAGATATTGAGC-3, SEQ ID NO: 9; LIG4 primer Exon2-1 reverse primer 5-AGAACCTTCAGTAGGAGAAGCACCAA-3, SEQ ID. NO.: 10; LIG4 primer Exon2-2 forward primer 5-CCTGGTGAGAAGCCATCTGT-3, SEQ ID. NO.: 11; LIG4 primer Exon2-2 reverse primer 5-GCCTTCCCCCTAAGTTGTTC-3). After electrophoresis on 1% agarose gel, the mutant analysis was confirmed by sanger sequencing of the PCR product in which the expression of LIG4 was confirmed through Et-Br staining.

(103) Experiment Result

(104) As a result of analyzing whether there is any mutation in the C8, C27, G833, and T1704 sites of LIG4 in 39 colon cancer patient tissues, mutations in LIG4 were analyzed in 9 colon cancer patient tissues (Table 6).

(105) TABLE-US-00006 TABLE 6 Human colorectal cancer tissue LIG4 (DNA ligase IV) mutation No. C8T C26T G833A T1704C 1 WT WT WT WT 2 WT WT WT WT 3 WT WT WT WT 4 WT MT WT WT 5 WT WT WT WT 6 WT WT WT WT 7 WT MT WT WT 8 WT WT WT WT 9 WT WT WT WT 10 WT WT WT WT 11 WT WT WT WT 12 WT MT WT WT 13 WT WT WT WT 14 WT WT WT WT 15 WT WT WT WT 16 WT WT WT WT 17 WT WT WT WT 18 WT WT WT WT 19 WT WT WT WT 20 WT WT WT WT 21 WT WT WT WT 22 WT WT WT WT 23 WT WT WT WT 24 WT WT WT WT 25 WT WT WT WT 26 WT WT WT WT 27 WT WT WT WT 28 WT WT WT WT 29 WT WT WT WT 30 WT WT WT WT 31 WT WT WT WT 32 WT WT WT WT 33 WT WT WT WT 34 WT WT WT WT 35 WT WT WT WT 36 WT WT WT WT 37 WT WT WT WT 38 WT WT WT WT 39 WT WT WT WT

Example 26: Analysis of Drug Efficacy in Colon Cancer Patient-Derived Cells in which a p53 Genotype is a Normal Type and an LIG4 Genotype is a Normal or Mutant Type

(106) In order to analyze the degree of drug efficiency of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, in the colon cancer patient-derived cells 11-CT-79558D (p53 WT/LIG4 MT), 11-CT-80464B (p53 WT/LIG4 MT) and 11CT-94575 (p53 WT/LIG4 WT), 13CT-78649B (p53 WT/LIG4 WT) in which a p53 genotype is a normal type and an LIG4 genotype is a normal or mutant type, four different types of human colon cancer patient-derived cells REBM (Renal Growth Basal Medium; 5% FBS, 1% penicillin/streptomycin) in which a p53 genotype is a normal type and an LIG4 genotype is a normal or mutant type, and cultured in a 60 mm plate at 110.sup.5 cells/well for 24 hours at 37 C. The cells were treated with 25 M and 50 M of Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a competitive drug PARP inhibitor. After 48 hours of culturing, the change of cell shapes was analyzed to confirm the drug efficacy.

(107) Experiment Result

(108) As a result of observing the degree of change in cell shape by treating the human colon cancer patient-derived cells in which a p53 genotype is a normal type and an LIG4 genotype is different with Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, and olaparib, which is a PARP inhibitor, using a microscope, it was observed that the cell shape for Compound B, which is a novel PARP/Tankyrase simultaneous inhibitor, died only in the colon cancer patient-derived cells 11-CT-79558D (p53 WT/LIG4 MT) and 11-CT-80464B (p53 WT/LIG4 MT) in which a p53 genotype is a normal type and an LIG4 genotype is a mutant type, and that there was no change in cell shape in olaparib, which is a PARP inhibitor, thus exhibiting drug reaction for apoptosis by Compound B only in case where a p53 is a normal type and an LIG4 is a mutant type (FIG. 20).

(109) From the foregoing, the specific portions of the present disclosure have been described in detail. Therefore, it is apparent to a person having ordinary skill in the pertinent art that such specific technology is merely a preferable embodiment, and the scope of the present disclosure is not limited thereto. Accordingly, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.