표적암치료를 위한 저분자 STAT3 저해제인 옥사디아졸계 ODZ10117의 개발

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의과학과, 2020. 8. 예상규.STAT3 is a transcription regulator involved in many intracellular functions, including cell proliferation, differentiation, survival, angiogenesis, and immune response. Persistently activated STAT3 is a promising target for a new class of anticancer drug development and cancer therapy, as it is associated with tumor initiation, progression, malignancy, drug resistance, cancer stem cell properties, and recurrence. Here, I discovered 3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole (ODZ10117) as a small molecule inhibitor of STAT3 and suggested that it may have an effective therapeutic utility for the STAT3-targeted cancer therapy. ODZ10117 targeted the SH2 domain of STAT3 regardless of other STAT family proteins and upstream regulators of STAT3, leading to inhibition of the tyrosine phosphorylation and transcriptional activity of STAT3. The inhibitory effect of ODZ10117 on STAT3 was stronger than the known STAT3 inhibitors such as S3I-201, STA-21, and nifuroxazide. Furthermore, I demonstrated the therapeutic efficacy of ODZ10117 by targeting STAT3. ODZ10117 suppressed the cancer cell migration and invasion, induced apoptotic cell death, and reduced tumor growth in both in vitro and in vivo models of breast cancer and glioblastoma. In addition, ODZ10117 suppressed stem cell properties in glioma stem cells (GSCs). To confirm these results, I demonstrated two different types of xenograft model. First, I have shown that extended the survival rate and reduced lung metastasis in models of breast cancer. Next, the administration of ODZ10117 showed significant therapeutic efficacy in mouse xenograft models of GSCs. In conclusion, I believe this study provides insight in to a promising therapeutic candidate for cancers by targeting STAT3.신호변환 및 전사활성인자 3 (STA3)는 많은 종양에서 과발현하고 있으며, 종양 미세 환경에서 STAT3는 다양한 경로를 통해 지속적으로 활성화되며, 일반적으로 지속적으로 활성화된 STAT3는 종양의 형성, 진행, 악성도, 재발 및 약물 저항성, 암 줄기세포 특성과 연관되어 있다. 따라서, STAT3는 암 치료에서 성공 가능성이 매우 높은 단백질로 새로운 종류의 항암제 개발과 암치료에 유망한 표적이다. 이 연구에서 STAT3의 소분자 억제제로서 3-(2,4-디클로로-페녹시메틸)-5-트리클로로메틸-[1,2,4]옥사디아졸 (ODZ10117)을 발견하였고 이는 STAT3 표적 암치료에 효과적인 치료 효용이 있을 수 있음을 시사하였다. 먼저, ODZ10117은 다양한 종류의 암, 특히 유방암과 신경교모세포종에서 STAT3 활성화를 효과적으로 억제하는 것을 확인하였으며, 흥미롭게도 다른 STAT 계열 단백질 및 STAT3 상위 신호전달계에 관계없이 STAT3의 SH2 도메인을 표적으로 하여 STAT3의 타이로신 인산화, 핵 내로의 이동 및 전사 활성을 억제하는 것을 확인하였다. 또한, STAT3에 대한 ODZ10117의 억제 효과는 STAT3 억제제인, S3I-201, STA-21 및 니프록사지드와 같은 잘 알려진 STAT3 억제제 보다 STAT3의 활성화 저해 능력이 뛰어났다. ODZ10117은 암세포의 이동과 침윤을 억제하고, 세포사멸을 유도하였으며, 종양의 성장을 감소시켰다. 이러한 결과를 확인하기 위해, 본 연구에서는 두가지 암 종류에 해당하는 이종 이식 모델을 연구하였다. 첫번째로, 유방암 모델에서 ODZ10117의 억제 효과는 종양형성을 억제하였으며, 마우스의 생존율을 높이고 폐 전이를 감소시키는 것을 확인하였다. 다음으로, ODZ10117의 투여는 신경 교종 줄기세포의 마우스 이종 이식 모델에서도 종양형성을 저해하고 생존율을 높이는 등 치료 효과를 보였다. 결론적으로, 새롭게 발굴한STAT3의 소분자 억제 화합물인 ODZ10117은 종양에서 STAT3의 활성화 억제를 통해 항암치료에 대한 새로운 치료 전략이 될 수 있음을 시사한다. 나아가, 종양 미세 환경에서 STAT3의 역할과 종양 미세 환경에서의 ODZ10117의 새로운 역할을 규명함으로써, 암세포 뿐만 아니라, 종양 미세 환경에서도 STAT3 저해제의 항암작용 역할을 기대할 수 있다.Introduction 1 Materials and methods 8 Results 20 Figures 34 Discussion 74 References 79 Abstract in Korean 86Docto

Modification of host and bacterial protein expression by Orientia tsutsugamushi invasion

학위논문(석사)--서울대학교 대학원 :치의학과 구강악안면 감염-면역학전공,2005.Maste

Analysis of Fusobacterium nucleatum antigens: identification of in vivo-induced antigens and functional analysis of GroEL, a heat shock protein

Objectives Periodontitis is one of the most predominant chronic inflammations of the oral cavity, and has been reported to be associated with systemic diseases. Among the periodontal pathogens, Fusobacterium nucleatum is one of the most predominant bacterium found in periodontitis and plays an important role for subgingival biofilm formation by mediating multiple interactions between early and late colonizers. Accumulating data have demonstrated an association between periodontal pathogens and cardiovascular diseases. And bacterial GroEL, a homologus protein of human heat shock protein 60, is represented as an additional risk factor for atherosclerosis progression. Despite the importance of F. nucleatum in human disease, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. The purpose of this study is to identify the F. nucleatum genes induced in vivo using in vivo-induced antigen technology (IVIAT) and to investigate whether GroEL of F. nucleatum induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE-/-) mice. Methods To identify specifically expressed F. nucleatum genes during infection, an immune-screening technique termed IVIAT was performed. An IPTG-inducible expression library of F. nucleatum was constructed using pET30 expression vector and F. nucleatum genomic DNA. Total 30,000 recombinant clones of a F. nucleatum genomic DNA expression library were reacted with pooled sera from 10 periodontitis patients pre-adsorbed against in vitro-grown F. nucleatum and Escherichia coli. The reproducibly reactive clones were selected and the inserted DNA sequences in expression vector were analyzed by sequencing to identify the genes. To verify in vivo-induction of genes, the expression of 10 selected genes among the IVIAT-identified genes were analyzed by real time RT-PCR after F. nucleatum was infected into a human epithelial cell line, HOK-16B cells. To investigate the role of F. nucleatum GroEL in the progression of atherosclerosis, recombinant GroEL (rGroEL) of F. nucleatum was produced and tested for its endotoxin decontamination. Invasion assay of F. nucleatum into an endothelial cells, HMEC-1, was performed and then intracellular bacteria were observed and quantified by confocal laser scanning microscopy and flow cytometry, respectively. Anti-F. nucleatum GroEL Ab in serum and F. nucleatum DNA in gingival crevicular fluid were analyzed in periodontitis patients and healthy subjects. rGroEL-treated HMEC-1 cells were analyzed for the expression of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, tissue factor (TF), and tissue factor pathway inhibitor (TFPI). rGroEL-induced foam cell formation, a hall mark of atherosclerotic lesion, was detected. And the monocyte adhesion to HMEC-1 and transendothelial migration activity were also tested. To explore the effect of F. nucleatum and rGroEL on atherosclerotic lesion progression, 10-week old ApoE-/- mice were fed high fat diet and mice were injected with live F. nucleatum (5 × 107 CFU/mouse), rGroEL (50 μg/mouse), or PBS once a week for 8 weeks. The atherosclerotic lesion in proximal aorta of the mice was detected and the risk markers (C-reactive protein (CRP), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and IL-6) of atherosclerosis were analyzed in serum. And the intracellular signaling pathway of GroEL in HMEC-1 was analyzed using specific inhibitors of MAPK and NF-κB and TLR4 siRNA knockdown system. Results For detection of in vivo-induced genes, 87 clones showed reproducibly the reactivity with pooled sera from 10 periodontitis patients. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. The expression of 10 selected in vivo-induced genes were verified to be increased in F. nucleatum after infection into HOK-16B cells, an epithelial cell line. F. nucleatum invaded the HMEC-1 cells and GroEL induced the expression of chemokines such as MCP-1 and IL-8 as well as cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. GroEL induced the activity of TF and reduced the activity of the TFPI. Foam cell formation was induced by GroEL. GroEL-injected ApoE-/- mice showed significant atherosclerotic lesion progression compared to control mice. Serum levels of risk factors for atherosclerosis such as IL-6, CRP, and LDL were increased in GroEL-injected ApoE-/- mice compared to control mice, whereas serum levels of HDL were decreased. There were significantly higher levels of anti-F. nucleatum GroEL Ab in serum and F. nucleatum DNA in gingival crevicular fluid in periodontitis patients than those of healthy subjects. The expression levels of GroEL-induced inflammatory mediators were mediated by activation of NF-κB and MAPKs through TLR4 signaling pathway. Conclusion In vivo-induced 32 immunogenic proteins of F. nucleatum could be used to screen antibacterial and anti-inflammatory compounds using a cell infection model. The ability of F. nucleatum GroEL to induce the expression of host factors that were involved in atherosclerosis progression in endothelial cells and ApoE-deficient mice models supports the association of periodontitis and systemic infection.Docto

리포솜 봉입이 약물의 흡수에 미치는 영향

학위논문(석사)--서울대학교 대학원 :약학과 약제학전공,1999.Maste