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Discovery of Autophagy Modulator using Image-Based High-Content Screening with Lipid Droplet Bioprobe : 지방방울 바이오프로브와 고효율 이미지 기반 스크리닝 시스템을 통한 자가포식 저분자 조절 물질 개발

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Authors
유빈
Advisor
박승범 교수님
Major
자연과학대학 화학부
Issue Date
2015-08
Publisher
서울대학교 대학원
Keywords
autophagyamyotrophic lateral sclerosis (ALS)neurodegenerative diseasesSeoul-Flour 44 (SF44)lipid droplet (LD) screeninghigh-content screening (HCS)autophagic fluxautophagy modulatorLC3p62mechanism of-action studymTOR-independent autophagystructure-activity relationship analysis (SAR)cellular ALS modelsmutant SOD1Alzheimer’s diseasetarget identification
Description
학위논문 (석사)-- 서울대학교 대학원 : 화학부 생화학전공, 2015. 8. 박승범.
Abstract
Autophagy is a major clearance and pro-survival pathway for recycling cytoplasmic components and for the removal of disease-causing aggregated proteins, organelles, and lipid droplets by lysosomal degradation [1]. Basal level of autophagy is essential for balancing the protein quality and degradation and recycling of cellular components while maintaining cell viability. The formation of intra-neuronal mutant protein aggregates is a key characteristic of several human neurodegenerative diseases such as Alzheimers disease and Amyotrophic lateral sclerosis (ALS). Therefore, protein degradation can enhance the clearance of misfolded and accumulated protein aggregates that have not been efficiently degraded due dysregulated autophagy. This enhancement of aggregated protein clearance may ameliorate neuro-degeneration disease [2]. Herein, we aimed to identify a novel small molecule autophagy modulator using our fluorescent bio-probe Seoul-Flour 44 (SF44) to monitor hydrophobic cellular lipid droplets and lipid metabolism, which can be mediated by the autophagy process. This lipid droplet (LD) screening method was applied to a phenotypic and cell image-based high-content screening (HCS) system in living cells, and it can discriminate the effects of chemical modulators on autophagic flux with cellular LD levels as a late-stage marker of autophagy [3]. As a first step, about 1400 library compounds were screened using SF44 to identify active lead compounds that can induce autophagy without causing cytotoxicity and off-target effects. The primary positive compounds were further tested by western blot analysis of LC3 lipidation and p62 to monitor autophagy. Then, mechanism of-action study of the hit compound confirmed that it stimulates mTOR-independent autophagy, and the hit compound was further investigated by a structure-activity relationship analysis (SAR) in search of a potent and promisingly active compound structure. We also discussed the possible implications of autophagy inducer in cellular ALS models that the autophagy induction mitigates neurodegeneration by acting directly on mutant SOD1 clearance, which can be potential therapeutic approaches for ALS and related conditions. This study will be informative for the growing field as research tools and developing drugs for autophagy-related diseases such as Alzheimers and ALS diseases, for which there have been many failures of target-based drug candidates in clinical trials. In-depth characterization of the compound properties including target identification can aid in the design of improved next-generation drugs for neurodegenerative diseases.
Language
English
URI
https://hdl.handle.net/10371/134923
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College of Humanities (인문대학)Korean Language and Literature (국어국문학과)Others_국어국문학과
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