(Microsoft PowerPoint \261\307\310\243\301\244)

Size: px

Start display at page:

Download "(Microsoft PowerPoint \261\307\310\243\301\244)"

온조 오
4 years ago
Views:

1 Efficient Drug creening model: 생리활성물질탐색및최신방법 권호정, 정혜진 연세대학교생명공학과 화학유전체학연구실

2 목 차 신약후보물질개발과생리활성물질탐색법 생리활성물질탐색의원리 생리활성물질탐색의최근기술 오토파지활성화합물의세포기반탐색

3 Drug discovery overview

4 creening phase Primary creening econdary creening igh Throughput creening Cell-based igh Content creening Identification of new drug targets Validation of hits Discovery of new leads ptimization of leads Development of candidates ew Drugs! Genomics Proteomics

5 생리활성물질탐색의접근법 Target-based screening Cell-based screening Target identification uman cell systems Target-directed screening (target hit) Cell/phenotype-based screening (biological hit) Lead optimization Lead optimization Preclinical and clinical tests Preclinical and clinical tests Drug Drug

6 AP 저해제의 target-based screening Assay principle 2 3 C 2 Alanine-7-amido-4-methylcoumarin (fluorogenic substrate) purified porcine AP 3 C 2 ubstrate degradation Detection of fluorescence T screening of AP inhibitors 3,000 natural products & chemical libraries him J, et al. Chem. Biol. 2003; 10 Multiwell plates Fluorescence microplate reader Data analysis

혈관신생저해제의 cell-based screening mall molecule library

screening Targets its 2 C3 Br 3C Br C3 2C Cl Br Br C 3a

screening (high dose-50 µg/ml) Br Br 250 compounds were

compounds were selected AP (3) DAC (3) A-LAP (2) IF-1α

(38) Curcumin A BetA RA12a PsamA MPA 6-TG Pupn BC

7 혈관신생저해제의 cell-based screening mall molecule library Cell/phenotype-based T screening Target-oriented screening Targets its 2 C3 Br 3C Br C3 2C Cl Br Br C 3a Br 3 Br - C C 2 C 2 Cl 2 C3 CF 3 3C CF 3 C3 Primary screening (high dose-50 µg/ml) Br Br 250 compounds were selected econdary screening (low dose-10 µg/ml) 46 compounds were selected AP (3) DAC (3) A-LAP (2) IF-1α (0) thers? (38) Curcumin A BetA RA12a PsamA MPA 6-TG Pupn BC Terpestacin 3,000 compounds from natural products and chemical library IF Tanshinone etc Kwon J, Curr. Drug Targets 2006; 7

8 생리활성물질탐색의원리 흡광 (Absorbance) 생물발광 (Bioluminescence) 형광 (Fluorescence) 형광공명에너지전달 (Fluorescence Resonance Energy Transfer; FRET) 생물발광공명에너지전달 (Bioluminescence Resonance Energy Transfer; BRET)

Mitochondrial reductase Living cells MTT (Yellow)

9 흡 광 원리 효소에의해생성된물질이특정파장의빛을흡수함으로써효소반응전후다른색을발색 MTT assay 세포의증식을측정하기위한표준비색분석법 (540 nm 의파장에서흡광도측정 ) Mitochondrial reductase Living cells MTT (Yellow) MTT-formazan (Purple) (µm) Anti-cancer drug

10 생물발광 원리 특정환경에서외부의빛에너지공급이없이독자적으로빛을발산하는현상 어떤유기화합물이효소의작용으로산화되면서그때방출되는에너지가빛에너지의형태로체외로 나오는현상으로일종의광화학반응 Luciferase assay Double readout for p53 transcriptional activity Firefly luciferase (FL), Renilla luciferase (RL)... p53 p53re FL CMV PM RL Firefly luciferase ATP + 2 Mg 2+ AMP + PP i + C 2 + Light (λ max = 560 nm) DA damaging agent (p53)-fl (CMV)-RL Luciferin xyluciferin +

11 형 광 원리 외부의특정파장의빛에너지를흡수한형광물질이그보다낮은에너지의장파장빛을다시방출하는현상 형광발생체 형광화합물 (FITC, Texas Red, Cy5, Cy3, DAPI) 형광단백질 (GFP, RFP) Tubulin immunostaining using FITC-labelled antibody ormal cell Taxol-treated cell FITC Excitation: 488 nm Emission: 530 nm uclear translocation of GFP-tagged FAT o stimuli Ionomycin Ionomycin + CsA

12 형광공명에너지전달 원리 서로다른형광을내는두개의물질이 10 nm 이내로가까워짐으로써그사이에공명에너지전달이 생겨각자의형광스펙트럼이달라지는현상 들뜬상태의공여체분자가수용체분자에에너지를전달하여수용체분자의전달된에너지양을측정 R 측정시스템 세포내칼슘이온농도측정시스템 FRET 442 nm 480 nm 530 nm CFP YFP xidized CFP YFP Reduced R FRET ratio (CFP/YFP ratio) 143B WT + tigmatellin chumacker PT, et al. J. Biol. Chem. 2007; 282

13 FRET + β-lactamase (β-lac) cell based assay taurosporine inhibition of beta-lactamase expression 460/528-emission ratio concentrations of staurosporine o β-lac expression β-lac expressing BLA-AM substrate Cytoplasmic Esterases 520 nm 409 nm 447 nm Cell membrane Cytoplasm 409 nm β-lactamase BLA substrate

14 FRET + β-lactamase (β-lac) cell based assay Ligand Ligand Gα Gβ Gγ Gs Gα Gβ Gγ Gq GPCRs Receptor AC PLC ATP camp PKA Ca 2+ C 2 nd messenger CREB P CREB CRE bla P EAT EAT EAT FATRE bla Blue Product Kinase Transcription Factor β-lactamase Green ubstrate CRE-bla

15 생리활성물질탐색의최근기술 고속다중스크리닝기술 (igh Throughput creening; T) 대량정보스크리닝기술 ( igh Content creening; C) 가상스크리닝기술 (Virtual creening) 케미컬칩스크리닝기술 (Chemical Chip creening)

16 igh Throughput creening (T) 정의 약효스크리닝의과정 ( 화합물의분주, 희석, 스크리닝성분혼합, 배양및검출, 스크리닝데이터의분석 및결과보고 ) 을신속하고효율적으로처리하기위해소형화및자동화된고속다중스크리닝시스템 Chemical genomics T combines two components : the chemistry and the targets. Compounds : In T, a useful library is generally considered to be between 100,000 and 1,000,000 compounds. Targets : The number of targets is clearly finite.

17 고속다중스크리닝기술 Identification of polyglutamine aggregation inhibitors by T protease GT-huntingtin fusion protein compound library (384 well format) DM control I Congo Red - Protease control II cleavage reaction Thio- DM + Protease filtration formation of aggregates immuno detection of aggregates image analysis and hit identification F8 inactive inactive inactive active

18 igh Content creening (C) 정의 세포내다양한표적에대하여살아있는세포를대상으로고해상도의형광영상을얻고, 이를컴퓨터 프로그램을이용하여시간적, 공간적결과로분석하여복합적인정보를스크리닝하는기술 C system Active caspase-3 3 staining in ela cells after staurosporin treatment igh content imaging

19 C workflow Automated Plate Delivery Auto-focus, Expose & Acquire Automated Image Analysis Analysis of Results Instantaneous Data Display Automatic Data torage

stats based on individual cells Low biological

information like area, shape, and texture o

20 igh Throughput vs. igh Content T C ne number per well (absorbance, fluorescence, luminescence, etc.) Multiple metrics per well, population means or stats based on individual cells Low biological content information igh biological content information like area, shape, and texture o individual cellular measurements Cell by cell identification and analysis allows subpopulations to be measured

21 가상스크리닝기술 원리 컴퓨터를이용하여대규모화합물라이브러리의데이터베이스를표적수용체에도킹시켜예상약효화합물을가상스크리닝 가상검색을위한도킹알고리즘 - AutoDock, DCK, FlexX, FLG, GLD, ICM, LUDI, LIDE, EUDC, EED, MM o KT, Bio-Medical cience 2005; 3

22 Chemical library space atural product space Drug space 6X10 6 Combinatorial Library space Drug-like space mall-molecule chemistry space 10 20~ ~64. Rose et al., Current pinion in Chemical Biology (2003)

Product Library 2 C C 2 CC C 2 C 2 CC C 2 C 2 C 2

C 2 2 CC C 2 C 2 2 CC C 2 2 CC C 2 C 2 C C C 2 C 2

2 2 C C C 2 2 CC C 2 2 C C C C 2 2 C C C Medicinal

9 12 14 16 18 20 10 C 2 C 3 3C 2 CC 2 2 C C C C 2C

2C 4 4 4 2 C 2 3C 3C 3C C 3C Cl 3C C 2C 3CC Cl 2 2

: 50,240 ICCB + 2 C 2 2 I 3C 2 2 C C C C 3C 2 2 2

23 Chemical library: Quantity or Quality? Chemical Library (Combinatorial Library) atural Product Library 2 C C 2 CC C 2 C 2 CC C 2 C 2 C 2 C 2 2 C C C 2 C 2 C 2 C CC C 2 C 2 2 CC C 2 C 2 2 CC C 2 C 2 2 CC C 2 2 CC C 2 C 2 C C C 2 C 2 C 2 C C C 2 2 C C C 2 C 2 C 2 2 CC C 2 CC 2 C C C 2 2 C C C 2 2 CC C 2 2 C C C C 2 2 C C C Medicinal plants Curcumin Cryptotanshinone omoisoflavanone C 2 C 3 3C 2 CC 2 2 C C C C 2C 2C 3C 2 3C Cl 2 A c C 3C C 3C e 2 2 CF 3.2C C 2 3C 3C 3C C 3C Cl 3C C 2C 3CC Cl C F 3C 3C 3C 3C ChemBridge Corp. : 50,240 ICCB + 2 C 2 2 I 3C 2 2 C C C C 3C C C Curcuma longa Microbial metabolites Radicicol Cl Acremonium sp. And more... alvia miltiorrhiza Terpestacin Embelisia chlamydospora Cremastra appendiculata MBA Curvularia sp.

24 Target-oriented library Protein information In silico screening mart chemical library 2 Cl 2 [1] [2] [3] [4] [5] Cl Cl 2 2 Chemical information [6] [7] [8] [9] [10] 2 2 [11] [12] [13] [14] [15] Cl [16] [17] [18] [19] [20] 3.6 million compounds (from Pharmo DB) <1,000 compounds Cost Time Quality

25 오토파지활성화합물의세포기반탐색 오토파지의정의 오토파지의생물학적기능 오토파지작용기전 오토파지유도화합물검색시스템

26 오토파지 (Autophagy) 오토파지? 자식 ( 自食 ) 작용 : 세포가자신의소기관을리소솜으로분해하는현상프로테아솜분해계와는대조되는비선택적인대규모분해계다이나믹한막변형 ( 격리막신장 오토파고솜형성 리소솜융합 ) 을수반오토파지는 apoptosis와는구분되는 caspase 비의존적 programmed cell death 로서세포의다양한고차기능조절에관여신규항암제개발을의한새로운표적으로주목받고있음 ormal cell Apoptosis Autophagy Galluzzi L, et al. Cell Death Differ. 2007; 14

오토파고솜외막에리소솜이융합하여오토리소솜이되면내막과내용물이리소솜의가수분해효소에의해소화됨 오토파지신호전달경로 오토파지는주로

27 오토파지작용기전 오토파지과정 Atg5 복합체와 LC3 가격리막에결합하게되면막의성장이개시됨 Atg5 복합체는오토파고솜의완성전후에막으로부터이탈하나 LC3 는결합된상태로존재함 오토파고솜외막에리소솜이융합하여오토리소솜이되면내막과내용물이리소솜의가수분해효소에의해소화됨 오토파지신호전달경로 오토파지는주로 PI3K-AKT-mTR 신호전달경로를통하여유도됨 mtr kinase 의활성화는오토파지의억제를야기하며이경로는많은암종에서활성화되어있음 Yoshimori T, BBRC 2004; 313 Rapamycin 은 mtr kinase 를저해하여오토파지를유도함

28 오토파지유도화합물검색시스템 오토파지활성세포기반검색계 Green: EGFP-LC3 Chemical libraries 37, 24 h EGFP-LC3 를일시적으로발현시킨 C7 세포에서 rapamycin 처리에의한오토파고솜의발달을확인 T Rapamycin 후보활성화합물의오토파지활성측정 A T YCG Rapamycin (A) MDC (Monodansylcadaverine) 염색 (B) Lysotracker 염색 B

29 igh content screening of autophage inducing agents LysoTracker staining h MY et al. BBRC 2008

30 Acknowledgements Chemical Genomics Lab. at Yonsei Univ. Dr. ye Jin Jung Yoon un Cho Ki yun Kim am ee Kim Beom eok Kim Junghwa Chang Yeon Jung Lee yung Keun Lee Juyearl Park BM, Thermo

36 Chap 20 : Conjugated Systems 20.1 Stability of Conjugated Dienes Diene : 2 개의 C=C 이중결합을가진화합물 C 1,4-Pentadiene 1,3-Pentadiene 1,2-Pentadiene (unconj

36 Chap 20 : Conjugated Systems 20.1 Stability of Conjugated Dienes Diene : 2 개의 C=C 이중결합을가진화합물 C 1,4-Pentadiene 1,3-Pentadiene 1,2-Pentadiene (unconjugated diene) (conjugated diene) (cumulated diene)