[보고서]Peroxiredoxin 활성억제를 통해 암세포를 선택적으로 사멸시키는 항암제의 개발

Peroxiredoxin 활성억제를 통해 암세포를 선택적으로 사멸시키는 항암제의 개발
Development of peroxiredoxin inhibitor as anticancer agent selectively killing cancer cells 원문보기

보고서 정보
주관연구기관	고려대학교 Korea University
보고서유형	1단계보고서
발행국가	대한민국
언어	한국어
발행년월	2011-09
과제시작연도	2010
주관부처	교육과학기술부 Ministry of Education and Science Technology(MEST)
등록번호	TRKO201300012717
과제고유번호	1345135485
사업명	미래기반기술개발
DB 구축일자	2013-08-26
키워드	레독스 밸런스.암세포.항암제.활성산소.퍼옥시레독신.선도 물질 최적화.redox balance.cancer cells.anticancer agent.reactive oxygen species.peroxiredoxin.lead optimization.
DOI	https://doi.org/10.23000/TRKO201300012717

초록 ▼

활성산소를 제거하는 효소인 Prx는 다양한 암세포에서 발현이 증가되어 있고, 항암제 개발에 있어 novel target으로 제시되었음. 본 연구진은 Prx 활성을 저해하는 유효물질을 개발하였음. 이 Prx 저해제가 활성산소증가를 매개로 하여 암세포의 선택적인 사멸을 유도하는 기전 규명과 hit-to-lead 및 lead optimization을 위한 target lead profile 도출 함
● Prx 활성을 저해하는 유효물질의 개발 가능 타당성을 입증
● 진행되고 있는 연구의 개선 방향 도출
● 국내 연구 인력분석을 통한 자문 위원회 형성
● 경쟁력확보를 위한 추진전략 도출을 위한 기획전략
● Prx 저해제의 선도물질 확보 및 최적화를 위한 target lead profile (약리, 약효. AMDE PK, 기초독성) 도출
● 유효물질의 최적화 단계에서 동물실험을 위한 potency 확인 및 향상 전략 수립
● Prx 저해제 유효물질의 선도 물질로의 도출 및 최적화를 위한 팀 구성과 추진일정을 경함

Abstract ▼

We will carry out the following studies to develop the inhibitor of Prxs that are able to eliminate ROS, and to reveal the mechanism that lead to ROS-mediated selective killing of cancer cells.
1. Project for research planning
● Analysis of appropriateness of research project for the development of Prx inhibitor
● Establishment of a research strategy
● Analysis of domestic research power and establishment of a research team
● Analysis of current patents and competitiveness
2. Main research project
① Lead discovery and optimization of Prx inhibitor
● Design and synthesis of structural derivatives of Prx inhibitor previouly identified as hit compound
● Measurement of Prx inhibitory activity
● Evaluation of in vivo toxicity of Prx inhibitor and pharmacokinetic parameters of Prx inhibitor
● Selection of optimized compound by analysis the cytotoxic effects
● Evaluation of anticancer effect of Prx inhibitor using xenograft animal model
② Investigation of ROS-mediated mechanism in selective killing cancer cells by Prx inhibitor
● Increase in cellular ROS level by Prx inhibitor
● ROS-mediated inhibition of cancer cell proliferation by Prx inhibitor
● Elucidation of the mechanism in cancer cell death by Prx inhibitor
● Investigation of the selective killing effects of Prx inhibitor on cancer cells

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
요 약 문 ... 4
SUMMARY ... 6
CONTENTS ... 8
목 차 ... 10
제 1 장 Peroxiredoxin 활성 저해 항암제 개발 기획사업의 개요 및 추진 체계 ... 12
제 1 절 연구의 필요성 ... 12
1. 경제적 •산업적 중요성 및 필요성 ... 12
2. Cancer 치료를 위한 분자 타겟으로서 Prx의 중요성 ... 17
3. 향후 전망 ... 30
제 2 절 선행연구 결과 ... 31
1. Prx 활성 억제제의 발굴 및 암세포 사멸효과 분석 ... 31
2. 다른 Prx 들을 고려한 C59의 선택적 효과 ... 35
3. 다른 oxidoreductase 들을 고려한 C59의 선택적 효과 ... 35
4. C59에 의한 Prx 활성 메커니즘의 규명 ... 36
5. 암세포 특이적 C59에 의한 세포사멸 효과 ... 37
제 3 절 연구추진체계 ... 40
1. 기획팀의 구성 ... 40
2. 연구추진계획 ... 41
3. 연구의 기대효과 ... 41
제 2 장 Peroxiredoxin 활성 저해 항암제 개발의 동향 ... 42
제 1 절 국내 •외 연구개발 동향 ... 42
1. 암세포 내 활성산소 증가 메커니즘을 통한 항암제 개발 ... 42
2. Prx 활성 저해제 개발 ... 44
3. 국내 • 외 관련 연구와의 차별성 ... 45
제 2 절 국내 • 외 시장 현황 및 전망 ... 46
1. 해당 신약개발 관련 시장조사(국내외 시장동향 포함) ... 46
2. 암세포 내 활성산소 증가 메커니즘을 통한 항암제 향후 전망 ... 48
3. Prx 활성 저해제 향후 전망 ... 48
제 3 절 특허 및 논문 분석 ... 49
1. 특허분석 ... 49
2. 논문분석 ... 50
제 3 장 Peroxiredoxin 활성 저해 항암제 후보 물질 개발 전략 수립 ... 52
제 1 절 분자 설계 및 구조결정 분석 ... 52
1. 현황분석 ... 52
2. 추진전략 ... 52
제 2 절 DMPK 지표 분석 ... 55
1. 현황분석 ... 55
2. 추진전략 ... 62
제 3 절 종양 약리 및 생물학적 분석 ... 67
1. 현황분석 ... 67
2. 추진 전략 ... 76
제 4 장 Peroxiredoxin 활성 저해 항암제 개발을 위한 연구 추진 로드맵 및 criteria ... 81
제 1 절 연구 추진 로드맵 ... 81
1. 총괄 로드맵 ... 81
2. 세부로드맵 (hit-to-lead 와 lead optimization) ... 82
제 2 절 선도물질의 선발 criteria 구성 ... 85
제 3 절 Decision tree의 결정 ... 87
제 4 절 자문회의 및 반영사항 ... 89
제 5 장 참고자료 및 문헌 ... 90

표/그림 (51)

표 Global cancer drugs market forecase, 2009-2021
표 Ten most common cancers by incidence
표 암세포 내에서 활성산소와 항산화 효소의 증가 및 균형
표 활성산소의 증가에 의한 암세포의 선택적 사멸작용
표 다양한 종류의 암 조직에서 Prx mRNA 발현증가
표 Prx family의 3개의 그룹에 따른 효소반응기전에 관한 모식도
표 사람의 6종 Prx 단백질들의 세포 내 발현 위치에 관한 모식도
표 Highly expressed Prx I in lung cancer. The expression levels of Prx I were compared by Western blotting between BEAS 2B and A549 and between normal (N) and cancer (C) tissues from the patients of squamous carcinoma (SQ) or adenocarcinoma (AC).
표 Percentages of positive Prx I (A) and Prx II (B) by immunohistochemistry in adenocarcinoma (ac), squamous cell carcinoma (sc), small cell carcinoma (smc) and nonmalignant control bronchi (co).
표 (A) Predominant Expressions of Prx I mRNA in Breast Cancer Tissue. Data show the transcript levels of Prx I. The BCRT II array (qRT-PCR) was used to determine the transcript levels of Prx I. Data were analyzed using the comparative CT method with the values normalized to β-actin level and expressed relative to controls. In parallel with each cDNA sample, standard curves were generated to correlate CT values using serial dilutions of the target gene. The y-axis represents the value of pg of DNA × 104. The BCRT II array consisted of five samples of normal breast tissue and 43 samples of breast cancer tissues from different individuals. Abbreviations: BCRT II, Human Breast Cancer qRT-PCR Array II. (B) Western Analysis of Prx I Protein Expressions in Malignant and Normal Tissues. Abbreviations: C, cancer (malignant); N, normal.
표 Protective role of cytosolic 2-cys Prx in the TNF-α-induced apoptotic death of human cancer cells. Control (CT2) and dominant negative mutant (DN10)-expressing HeLa stable cell lines. Cells treated with TNF-α (10 ng/ml) and cycloheximide (CHX) for the indicated times (A-B, D-F) or TRAIL (C). (A) Expression of cytosolic 2-cysPrx DN enhances ROS production. (B) TNF-α-mediated apoptotic death enhanced by expression of cytosolic 2-cysPrx DN. (C) TRAIL-induced apoptotic death is enhanced by expression of cytosolic 2-cysPrx DN. (D-F) Activation of caspase-8-Bid pathway is regulated by cytosolic 2-cysPrxs; (D) Induction of caspase-8 activity in CT and DN cells. (E) Cytochrome c release in CT and DN cells. (F) Induction of caspase-3 activity in CT and DN cells.treated
$Knockdown of Prx II enhances DNA damage-induced cell death. A, knockdown of PrxII expression by specific siRNAs. An siRNA targeting firefly luciferase is used as a control (siCon). The cells were treated with increasing concentrations of either etoposide (ETO) for 24 h. B, enhancement of cell death by PrxII knockdown in doxorubicin (DOX)-treated HeLa cells. C and D, enhancement of cell death by PrxII knockdown in HCT116 and U2OS cancer cells treated with 20 uM etoposide for 24 h. The knockdown of Prx II expression by siRNA is shown. Data in the graph are means ± S.D. of the percent of PIcells in the sub-G0 fraction (n=3; *, p<0.005). IB, immunoblot. (from Lee etal2010)$ 표 Knockdown of Prx II enhances DNA damage-induced cell death. A, knockdown of PrxII expression by specific siRNAs. An siRNA targeting firefly luciferase is used as a control (siCon). The cells were treated with increasing concentrations of either etoposide (ETO) for 24 h. B, enhancement of cell death by PrxII knockdown in doxorubicin (DOX)-treated HeLa cells. C and D, enhancement of cell death by PrxII knockdown in HCT116 and U2OS cancer cells treated with 20 uM etoposide for 24 h. The knockdown of Prx II expression by siRNA is shown. Data in the graph are means ± S.D. of the percent of PIcells in the sub-G0 fraction (n=3; *, p<0.005). IB, immunoblot. (from Lee etal2010)
표 다양한 종류의 암조직이나 세포에서 Prx I 또는 II 발현 억제에 의한 radiation sensitivity 증가
표 (A) Prx I antisense reduces expression of Prx I protein in human lung tumor cell lines. Prx I proteins of wild-type cells (untransfected A549 and H1299), vector-transfected, and Prx I antisense-transfected A549 and H1299 cells were measured by Western blot analysis. β-actin was used to calculate relative Prx I expression. (B) Prx I antisense significantly increased radiation sensitivity of A549 and H1299 cancer cells. Wild-type, vector-transfected, and Prx I antisense-transfected cells were irradiated with 0, 3, 6, and 9 Gy and survival curves determined by colony-forming assay. A549 and H1299 cells with stable transfection of Prx I antisense were more radiosensitive than untransfected or vector-transfected cells. (C) Prx I antisense reduced pulmonary metastasis of A549 and H1299 xenografts. Pulmonary metastasis incidence of indicated xenografts after animals were killed when tumors reached > 4 cm3 in size. Results of distant metastasis were mean values of three independent experiments (3 animals per experiment, 9 animals in total for each cell type). Values on Y-axis are percentage of mice that developed lung metastasis.
표 (A) Western analysis of PrxII in PrxII/siRNA-transfected MCF+FIR3 radioresistant cells. (B) Clonogenic survival assays for MCF+FIR3 and PrxII/siRNA-treated MCF+FIR3 breast cancer cells. ●, radiation-resistant MCF+FIR3 cells. ■, PrxII/siRNA-treated MCF+FIR3 resistant breast cancer cells. Clonogenic survival was calculated. The dose-modifying factors were calculated from the radiation doses corresponding to 10% survival.
표 (A) Inhibition of Prx II expression in Prx II antisense oligomer (AS1)-transfected 11A cells. Expression of Prx II was detected by Western blotting after transfection with AS1. Lane C, cells transfected with no DNA; Lane AS1, cells transfected with AS1. β-actin was run as a control. (B) Radiation sensitization of 11A cells after transfection with an antisense Prx II expression plasmid (pPrxII/AS). 11A cells were transfected with 0.025 (■), 0.05 (▲), and 0.1 mg/ml (▼) pPrxII/AS. The cells were then irradiated with 0.5, 1, 2, and 4 Gy. Cell survival was measured by a clonogenic assay. As a control, the cells were transfected with 0.05 mg/ml of sense Prx II expression plasmid (pPrxII/S; ●).
표 IC50 of compounds on peroxidase activity of Prx I.
표 1. Prx I inhibitor (C59) induces cell death in A549 human lung cancer cells. A, Cell proliferation was measured by WST -1 assay at 48 h after treatment of 10 uM chemicals . B, Time-dependent cytotoxicity induced by 10 uM C59 in A549 cells. Viable cells were measured by WST-1 assay at the indicated times after treatment of 10 uM C59. At the indicated times after treatment of 10 uM C59 compound, cellular ROS level (C) and cell death (D) were analyzed by flow cytometry using DCF-DA staining (C) and propidium iodide (PI)-Annexin V double staining (D) methods. Each data point is expressed as the mean ± SEM of triplicates. Key: *, P < 0.05, as compared with the vehicle control.
$Effects of C59 on mitochondria-mediated apoptotic signaling. After Incubation of 10 μM C59 for indicated times in A549 cells, Cell lysates were prepared to measure alteration of ΔΨm and cardiolipin oxidation. A, Comparison of oxidative damage to cardiolipin by 10 μM C59 in A549 cells measured by flow cytometry using NAO. M1 indicates subpopulation of cells that lost NAO signal due to cardiolipin oxidation. B, Preferential induction of mitochondrial transmembrane potential loss by 10 μM C59 in A549 cells detected by flow cytometry using TMRE. M1 indicates subpopulation of cells that lost transmembrane potential. C, Cytochrome C release from mitochondria was analyzed by immunoblot analysis using antibody to Cytochrome C with the cytosolic fraction seperated from A549 cell lysates. Loading control of each cytosolic fraction was used to PrxI. Caspase-3 (D) and Caspase-9 (E) activity in cell lysates were measured the rate of fluorescent light according to degradation of the synthetic substrate DEVD-AMC or LEHD-AFC.$ 표 Effects of C59 on mitochondria-mediated apoptotic signaling. After Incubation of 10 μM C59 for indicated times in A549 cells, Cell lysates were prepared to measure alteration of ΔΨm and cardiolipin oxidation. A, Comparison of oxidative damage to cardiolipin by 10 μM C59 in A549 cells measured by flow cytometry using NAO. M1 indicates subpopulation of cells that lost NAO signal due to cardiolipin oxidation. B, Preferential induction of mitochondrial transmembrane potential loss by 10 μM C59 in A549 cells detected by flow cytometry using TMRE. M1 indicates subpopulation of cells that lost transmembrane potential. C, Cytochrome C release from mitochondria was analyzed by immunoblot analysis using antibody to Cytochrome C with the cytosolic fraction seperated from A549 cell lysates. Loading control of each cytosolic fraction was used to PrxI. Caspase-3 (D) and Caspase-9 (E) activity in cell lysates were measured the rate of fluorescent light according to degradation of the synthetic substrate DEVD-AMC or LEHD-AFC.
표 Prx I, II, III, IV들의 peroxidase 활성에 대한 C59의 억제효과 비교
표 The Mechanism for inhibitory effect of peroxidase activity of Prx I by C59. A, 12.5 uM Prx I was incubated with 5 mM C59 in 20 mM Hepes (pH7.0) for 5 min prior to addition of 50 uM H2O2 for 30 min at 30oC and then reacted with 20 mM NEM and non-reducing sample loading buffer for 1 hr at RT. The oxidation state of Prx I was determined by immunoblot analysis with antibody specific to Peroxiredoxin I. B, After completely oxidation of 1 uM Prx I by 50 uM H2O2 for 5 min, H2O2 was eliminated by catalase at 25oC for 10 min. Oxidized Prx I and 5 mM C59 were incubated with the reaction mixture containing 3 uM yTrxI ,1.5 uM yTR, 200 uM NADPH for the indicated times at 30oC. The oxidation state of Prx I was determined by immunoblot analysis after incubation with 20 mM NEM and non-reducing sample buffer for 1 hr at RT.
표 Preferential induction of cell death and ROS accumulation by C59 in oncogenetically transformed cells. (A) Dese-dependent cell death induced by C59 in T80 and T80H cells. Cells were treated with the indicated concentration of C59 for 72 hr and viable cells were measured by WST -1 assay. (B) Time-dependent cell death induced by C59 in T80 and T80H cells. Cells were treated with C59 (50 uM) for indicated times and viable cells were measured by WST -1 assay. (C) At the 96 h after treatment of C59 (50 uM), cellular ROS level in T80 or T80H cells was analyzed by flow cytometry using DCF-DA staining. (D) Comparison of ROS in T80 cells and T80H cells induced by C59 (50 uM). Each data is expressed as the mean ± SEM of triplicates. Key: *, P<0.05, as compared with the T80 group.
표 C59 처리 후 ROS의 증가에 의한 cancer cell의 세포사멸
표 ROS 증가를 기전으로 개발되는 항암제
표 Arsenic trioxide 임상시험 목록
표 Phenylethyl isocyanate 구조와 임상시험 목록
표 2-Methoxyestradiol 구조와 임상시험 목록
표 Targeting cancer cells through ROS-mediated mechanism
표 약 카데고리별 시장 점유율
표 특허정보 분석 Aureka
표 Prx 관련특허의 연도별 출원/등록 건수 및 등고선 맵
표 논문 정보 분석 Scopus
표 Prx 관련 논문 연도별 게재 건수 및 등고선 맵
표 Prx I과 C59의 docking 결합 모델
표 분자모델링 프로그램
표 Prx 활성 저해제 선도물질 도출 전략
표 Drug-likeness 물질의 특성
표 신약개발을 위한 활성과 drug-like property의 상관관계
표 신약개발을 위한 연구체계
표 신약개발시 DMPK의 중요성
표 Intra- and inter day accuracy and precision of C59 spiked in rat plasma
표 Stability of C59 spiked in rat plasma (n = 3)
표 Mean plasma concentration-time curves of C59 following IV injection at a dose of 2 mg/kg and oral administration at a dose of 10 mg/kg and 20 mg/kg in rats. Bars represent the SD (n=3-6).
표 C59가 liver microsome을 이용한 대사안정성 실험에서 모두 빠르게 대사
표 Pharmacokinetic parameters of C59 following IV and oral administration to rats (n=3-6).
표 LMT163는 liver microsome을 이용한 대사안정성 실험에서 모두 빠르게 대사
표 C59에 의한 세포사멸이 암세포인 T80Ras 세포에서 민감하게 증가되는 효과
표 C59에 의한 세포 내 활성산소 축적이 암세포인 T80Ras 세포에서 민감하게 증가
표 C59에 의한 미토콘드리아 막의 cardiolipin 산화 (A)와 막전압의 변화 (B)가 암세포인 T80Ras 세포에서 민감하게 증가되는 효과
표 C59에 의한 T80Ras 세포 내 활성산소의 축적 (A), 미토콘드리아 막의 산화 (B), 미토콘드리아 막전압의 붕괴 (C), 이에 따른 세포사멸 (D) 등이 항산화제에 의해 억제되는 효과
표 동물모델에서의 C59 와 LMT163 항암 활성 효능(A) 및 실험동물의 체중 변화(B)

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총연구비 (DetailSeriesProject) :	-
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Peroxiredoxin 활성억제를 통해 암세포를 선택적으로 사멸시키는 항암제의 개발
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