초록 ▼

1) 케미컬 라이브러리를 스크리닝에 의한 NUAAP 발현 억제 Hit 화합물 도출 (3종)
- 케미컬 라이브러리 총 14,500 약물을 대상으로 HTS 스크리닝을 수행하였음.
- 암세포주, 정상세포주에 대한 약물의 반응, NUAAP 리포터 분석, NUAAP mRNA양 조사.
- NUAAP 발현억제, STAT3 단백질 분해 및 항종양 효능 약물 D6, D7, D14를 Hit 화합물로 발굴.
2) Hit 화합물 유도체 스크리닝에 의한 선도 물질 도출 (2종)
- Hit D7 유도체를 대상으로 최적화 수행을 통

1) 케미컬 라이브러리를 스크리닝에 의한 NUAAP 발현 억제 Hit 화합물 도출 (3종)
- 케미컬 라이브러리 총 14,500 약물을 대상으로 HTS 스크리닝을 수행하였음.
- 암세포주, 정상세포주에 대한 약물의 반응, NUAAP 리포터 분석, NUAAP mRNA양 조사.
- NUAAP 발현억제, STAT3 단백질 분해 및 항종양 효능 약물 D6, D7, D14를 Hit 화합물로 발굴.
2) Hit 화합물 유도체 스크리닝에 의한 선도 물질 도출 (2종)
- Hit D7 유도체를 대상으로 최적화 수행을 통해 항종양 활성이 5배 이상 향상된 선도물질 도출.
- 화합물 D629, D1237이 폐암 세포주 H1703에서의 NUAAP 발현 억제 및 STAT3 분해촉진, 세포사멸 유도 및 우수한 세포성장 억제 효능 확인하였음.
3) 선도물질의 NUAAP 발현 억제 기전 연구
- 선도물질은 in vitro/in vivo 모델에서 단백질 ubiquitination 증가에 의한 ER stress를 증가시켜 NFAT2 발현 감소, NUAAP mRNA 감소 및 세포사멸 유도함을 확인하였음.
4) 선도물질 D629 및 D1237의 in vitro/in vivo 항종양 활성 평가
- 다양한 폐암 세포주의 세포성장 억제를 보였음 (0.2-1.0 μM)
- EGFR 저해제 gefitinib/Erlotinib에 내성 폐암 PDX인 YL05와 YL08에 우수한 성장 억제 효능 있음.
- in vivo H1703 폐암세포주 xenograft 모델에서도 우수한 항종양활성을 나타내었음 (32%, 10 mg/kg)
5) 선도 물질 D629의 약동력학 평가
- 선도 후보물질 D629에 대해 in vitro/in vivo PK, microsomal stability, Cyb 약물 상호작용 저해 실험을 수행하였음.
(출처:요약서 3p)

Abstract ▼

NUAAP (FLJ25416, human Noxin, hNoxin, C11Orf82) shares 56% homology with mouse noxin. NUAAP was identified as a cancer-related protein which is highly expressed in colorectal and lung cancer tissues. The hNoxin gene (Genbank NM_145018) has six exons, including a translation start site in the third e

NUAAP (FLJ25416, human Noxin, hNoxin, C11Orf82) shares 56% homology with mouse noxin. NUAAP was identified as a cancer-related protein which is highly expressed in colorectal and lung cancer tissues. The hNoxin gene (Genbank NM_145018) has six exons, including a translation start site in the third exon, and encodes a 998-amino acid polypeptide. NUAAP contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. We characterized that NUAAP expression is induced by UV and that NUAAP is involved in anti-apoptotic function. Therefore, we named it NUAAP, Noxin-like UV-inducible anti-apoptotic protein, suggesting as a therapeutic target of lung cancer. In the previous study, we revealed transcription of NUAAP is regulated by NFAT2 and NUAAP stabilize STAT3 in lung cancer. In addition, HTS screening system to search inhibitors of NUAAP transcription using luciferase reporter gene under NUAAP promoter was established for the development of anti-cancer drugs.
The final goal of this study is to generate lead compounds to inhibit transcription of NUAAP and induce apoptosis of lung cancer cells. Chemical libraries should be screened using reporter system containing NUAAP promoter. Moreover, The structural optimization of hits will be carried out to improve anti-cancer efficacy in vitro and in vivo.
First, we obtained 14,500 chemicals in five different chemical libraries from KRICT, D group and L group and purchased from Sigma-Aldrich using established screening methods. We screened chemicals based on 1) the effect on growth inhibition of cancer cells, 2) the effect on growth inhibition of normal cells, 3) inhibition of NUAAP transcription using reporter assay of luciferase under NUAAP promoter and 4) the effect on expression of NUAAP mRNA and STAT3 protein. After large scale screening, we obtained three candidates compounds for Hits.
Then, we tested hit candidates compounds for the effect on the expression of dose-dependent and time-dependent inhibition of NUAAP mRNA and STAT3 and its target genes in various lung cancer cells. In addition, induction of apoptosis of cancer cells after treatment with the compound. Because NUAAP knockdown induces growth inhibition of cancer cells that are resistant to EGFR inhibitor such as Gefitinib and Erotinib, lung patient-derived xenograft cells (PDX) YL05 and YL08 were employed for as TKI-resistant cells. Hit compunds D7, D6 and D14 were satisfied with the criteria including inhibition of growth of YL05 and YL08 compared with that of Gefitinib or Eroltinib. D7 demonstrated 31% tumor growth inhibition with administration of 20 mg/kg in H1703 xenograft model.
The 30 derivatives of D7 were synthesized and provided for further screening by D group. We screened them for the effect on the inhibition of expression of NUAAP mRNA and STAT3. Compounds D629 and D1237 showed significant growth inhibition of most lung cancer cells with GI50 0.2~1.0 uM. Compound D629 and D1237 also induced apoptosis of various lung cancer cells, which was demonstrated by Western blot analysis. They showed strong growth inhibition of EGFR inhibitor-resistant YL05 and YL08 cells, suggesting significant improvement in growth inhibition of lung cancer cells. Similar molecular mechanism of NUAAP mRNA inhibition and STAT3 suppression and apoptosis induction was demonstrated in presence of Compound D629 and D1237. Therefore, we selected D629 and D1237 as candidates for lead compounds. Next, in in vivo xenograft assay using H1703 model, D629 showed 32% TGI with administration of 10 mg/kg. In addition, in vitro/ in vivo PK analysis of D629 was carried out at the the Bioevaluation center at KRIBB.
To understand how our compounds induce suppression of NUAAP mRNA expression, we studied action mechanism of D7 and its derivatives, D1237 and D629. Fortunately, we found fluorescent D7/D1237 localized at ER of cells and activates ER stress signaling, resulting in increasing expression of GRP and CHOP and phosphorylation of JNK. Then, we established in vitro and in vivo system to detect activation of ER stress using ERSE (ER response element)-luciferase reporter system. We tested D7, D629 and D1237 for activation of ER stress using reporter assay and in vivo bioluminescence imaging assay. As expected, they definitely activated ER stress when treated cancer cells, resulting in increase of luciferase activity and bioluminescence in tumor. The degree of ER stress activation of the compounds is correlated with growth inhibition of lung cancer cells. Because ER stress is activated by accumulation of ubiquitinated proteins through the inhibition of proteasomal pathway. We clearly observed that the ubiqutination of proteins significantly increased by the treatment of cells with either D7 or D1237, suggesting involvement in the inhibition of proteasomal degradation. We also attempted to identify target molecules of D1237 using chemical probes. After conjugating with biotin containing molecule, the affinity-based separated target molecules were identified by pull-down assay, SDS PAGE and mass analysis. Now, we are investigating the role of the tentative target molecule of D1237.
In summary, we screened chemical library of 14,500 compounds to search inhibitors of NUAAP expression. After selecting D7, D6 and D14 as hit compounds, we further optimized the structure of D7 and screened compounds for in vitro and in vivo efficacies of anti-cancer activity in lung cancer. D629 and D1237 were chosen as lead compounds to suppress NUAAP mRNA expression and STAT3 expression. D629 and D1237 are effective to inhibite growth inhibition of various lung cancer cells. Moreover, D629 and D1237 demonstrated significant efficacy to suppress growth of EGFR TKI-resistant PDX cells. In vivo anti-tumor efficacy and pharmacokinetic studies of D629 were carried out. We also revealed that D629 and D1237 activated ER stress signaling via accumulating ubiquitinated proteins and inhibit NFAT2 expression leading NUAAP mRNA downregulation, thereby inducing apoptosis of lung cancer cells.
(출처:SUMMARY 10~12p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 10CONTENTS ... 13목차 ... 14제1장 연구개발 과제의 개요 ... 15 제1절 NUAAP 발현 조절 약물 개발의 개요 ... 15 제2절 맞춤의료의 필요성 ... 16 제3절 표적 항암제 개발의 필요성 ... 17 제4절 암 치료 신규 타겟 유전자의 중요성 ... 17 제5절 NUAAP의 치료 타겟 항암제 개발의 필요성 ... 18제2장 국내외 기술개발 현황 ... 20 제1절 신약 개발 현황 ... 20 제2절 항암제의 개발 동향 ... 21 제3절 국내 표적 항암제 개발의 동향 ... 23 제4절 암 치료 타겟 유전자 ... 24 제5절 NFAT2의 암관련성 및 타겟으로의 중요성 ... 24 제6절 유전자 STAT3 및 NFAT2/STAT3 관련성 ... 26제3장 연구개발 수행 내용 및 결과 ... 28 제1절 실험재료 ... 28 제2절 실험방법 ... 29 제3절 결과 및 고찰 ... 34제4장 목표달성도 및 관련분야에의 기여도 ... 63제5장 연구개발 결과의 활용계획 ... 65 제1절 연구개발결과의 활용방안 ... 65 제2절 기대 성과 ... 66제6장 연구개발과정에서 수집한 해외과학기술정보 ... 67 제1절 NUAAP의 간암관련성 ... 67 제2절 STAT3와 DNA damage repair 관련성 ... 68 제3절 DNA damage 반응 단백질과 암 치료 타겟 ... 68제7장 연구시설·장비 현황 ... 70제8장 참고문헌 ... 71끝페이지 ... 74