chemical genetics
DESCRIPTION
OPSS. Chemical Genetics. --------Exploring the intersections between chemistry and biology. Supervisors: Prof. Zhen Yang Prof. Jiahua Chen Report: Jing Xiang( 向晶 ) 2007-6-8. Contents. Reverse Chemical Genetics and TOS Forward Chemical Genetics and DOS - PowerPoint PPT PresentationTRANSCRIPT
Chemical Genetics
Supervisors: Prof. Zhen Yang Prof. Jiahua Chen
Report: Jing Xiang(向晶 )2007-6-8
--------Exploring the intersections between--------Exploring the intersections betweenchemistry and biologychemistry and biology
OPSSOPSS
2
ContentsContents
Reverse Chemical Genetics and TOS
Forward Chemical Genetics and DOS
High Throughput Screening
Conclusion
3
To Inhibit a Known Protein?
Related to Virus( 病毒 ), Cancer or other diseases
Protein A How to inhibit it?
To improve the activity
To improve the selectivity
To design a inhibitor
Protein A with Known Inhibitor
Protein A without Known Inhibitor
TOS
4
TOS (Target Oriented Synthesis)
Focused Library (library=a collection of compounds)
Solid Phase Synthesis or Liquid Phase Synthesis
Targets: 1. Leading Compounds Natural Products Drug Candidates 2. Target Proteins
Spaller, M. R.; Burger, M. T.; Fardis, M.; Bartlett, P. A. Curr. Opin. Chem. Biol. 1997, 1, 47.Breinbauer, R.; Vetter, I. R.; and Waldmann, H. Angew. Chem. Int. Ed. 2002, 41, 2878
Schreiber, S. L. Science 2000, 287, 1964.
To synthesize a collection of compounds based on a target
5
TOS Based on Natural ProductsTOS Based on Natural Products
1. diversity2. new structure3. biological relevance
Natural products, based on their evolutionary selection, serve
as “biologically validated” starting points for library design.
----------Waldmann(Angew. Chem., Int. Ed. 2001, 41,
307 )
Antimitotic reagent
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
6
TOS Based on Natural ProductTOS Based on Natural Product
Epothilones
their mechanism of action against tumor cells has been attributed to the binding and stabilization of microtubules( 微管,由微管蛋白组装而成 ).
Nicolaou, K. C. et al. Angew. Chem. Int. Ed. Engl. 1998, 37, 2014
7
TOS Based on Natural ProductTOS Based on Natural Product
The Olefin Metathesis Approach of Epothilone A
Yang, Z.; He, Y.; Vourloumis, D.; Vallberg, H.; Nicolaou, K.C. Angew. Chem. Int. Ed. Engl. 1997, 36, 166
OHC
COOH
O OTBS
HO
O
COOH
OTBS
HO
O OTBSO
O
N
SHO
OOTBS
O
N
S
HO
O OH O
O
N
S
O
OH
N
SDCC, 4-DMAP
(45% overall yieldfrom B)
Ru
PCy3
PCy3
PhCl
Cl
A
B
C
50%
1. CF3COOH (20 voI.%), CH2CI2
2. mCPBA, benzene
(98%)
(55%)
LDA. THF
4
56
epothilone A
8
TOS Based on Natural ProductTOS Based on Natural Product
Retro-synthetic Analysis of TOS
K. C. Nicolaou, N. Winssinger, J. Pastor, S. Ninkovic, F. Sarabia, D. Vourloumis, Z. Yang, T. Li, P. Giannakakou, E. Hamel, Nature, 1997, 387, 268
O
O O
Me
N
SMe
OH
Me
HO
O
O
O O
Me
N
SMe
OR
Me
HO
O
O O
Me
N
SMe
OR
Me
HO
O
O
H
O
O
OH
OOR
OH
Me
N
SMe
EpoxidationMetathesis
EsterificationAldolcondensation
1 2
3
4
5
6
9
TOS Based Natural ProductTOS Based Natural Product
3 × 3 × 5 × 4 = 180
K. C. Nicolaou, N. Winssinger, J. Pastor, S. Ninkovic, F. Sarabia, D. Vourloumis, Z. Yang, T. Li, P. Giannakakou, E. Hamel, Nature, 1997, 387, 268
Building Blocks
R
O
H
O
O
OH
O
OH
Me
R3OH
N
SMe
OH
N
SMe
OH
N
SPh
OH
N
SMe
OH
N
O
OH
OOTBS O
OH
OOTBS O
OH
OOTBSR2
O
H
O
O
H
O
O
H
O
10
TOS Based on Nature ProductsTOS Based on Nature Products
Nicolaou, K. C. and Snyder, S. A.. Classics in Total Synthesis II: More Target, Strategies, Methods. WILEY-VCH GmbH & Co. KGaA. 2003:Chapter 7
Structure Activity RelationshipStructure Activity Relationship Better Activity
11
TOS Based on Drug CandidatesTOS Based on Drug Candidates
1VEGFR2/3 inhibitor
Not specific
Specific inhibitorVEGFR2
IC50=0.25nm
Christian Peifer et al. J. Med. Chem. 2006, 49, 1271-1281
Better Selectivity
12
TOS Based on Target Protein StructureTOS Based on Target Protein Structure
Hugo Kubinyi. Curr. Opin. in Drug Disc. & Dev. 1998, 41:4
Inhibitor designed for HIV protease
based on the protein’s crystal
structure
Active Center
N N
O
HN
NH
OHNH
HN
HO
OH
O
OO
OH
HN
OO
O
HN
O
O
O
NH
O
NH
NH
NH
Ile 50 Ile 50'
Gly 48 Gly 48'
Asp 30 Asp 30'
Asp 25 Asp 25'
O
HIV Protease( 艾滋病毒蛋白酶 )
13
ContentsContents
Reverse Chemical Genetics and TOS
Forward Chemical Genetics and DOS
High Throughput Screening
Conclusion
14
Human Genome (Human Genome ( 人类基因组人类基因组 ))
~30,000 Genes~100,000 Proteins fewer than 500 proteins have fairly well-elucidated biological functions
J. Drews, Nature Biotech. 1996, 30, 97-108A.L. Hopkins, C.R. Groom, Nat. Rev. Drug Discov. 2002, 1, 727-730
For Research:To find regulators for all the proteins
5,000-10,000 drugable targets within the human genome Only fewer than 500 targets are well explored
For Drug Discovery:To find new target proteins, More leading compounds
Forward Chemical Genetics + DOS
15
Small Molecules and Targets Proteins Identified in FCGSmall Molecules and Targets Proteins Identified in FCG
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
16
How to Find New Target Protein and New Leading Compound
Mammalian cellsHomology( 同源性,相似性 ) to humans Physiological context
Diverse Library ???
organisms
Produceeggs
96-well plate, one well one compound
17
Resource of Diverse Library
Prospecting Library
Solid Phase Synthesis
Structure Diverse Molecules:Diversity Generating Processes1. Appendage Diversity: Benzopyrans, Shikimic Acid 2. Stereochemical Diversity: [3+2], D-A3. Skeletal Diversity: a). Differentiation Process b). Folding Process
Burke, M. D.; Schreiber, S. L. Angew. Chem. Int. Ed. Engl. 2004, 43, 46.Schreiber, S. L. Science 2000, 287, 1964.
DOS (Diversity Oriented Synthesis)
Commercial LibrariesAvailable: >1500 compound libraries (data of 2005) Not Available: Pharmaceutical companies
18
DOS – Appendage Diversity Generating ProcessDOS – Appendage Diversity Generating Process
Nicolaou, K. C. et al. J. Am. Chem. Soc. 2000, 122, 9939-53, 9954-67, 9968-76.
19
DOS – Stereochemical Diversity Generating ProcessDOS – Stereochemical Diversity Generating Process
Catalytic Asymmetric [3+2] Cycloaddition of Azomethine Ylides.
Chen, C.; Li, X.; Schreiber, S. L. J. Am. Chem. Soc. 2003, 125, 10174.
Stereochemical diversification of up to 4 tetrahedral centers
20
DOS – Skeletal Diversity Generating ProcessesDOS – Skeletal Diversity Generating Processes
Differentiating Process &
Folding Process
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46-58Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
21
DOS – Skeletal Diversity Generating ProcessesDOS – Skeletal Diversity Generating Processes
Differentiating Processes
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46-58Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
A
B
C
22
DOS – Skeletal Diversity Generating ProcessDOS – Skeletal Diversity Generating Process
Folding Processes
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
cis-enedione intermediate
23
Forward and Reverse Chemical GeneticsForward and Reverse Chemical Genetics
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
How to determine the effect of the compounds in Libraries?
24
ContentsContents
Reverse Chemical Genetics and TOS
Forward Chemical Genetics and DOS
High Throughput Screening
Conclusion
25
High Throughput Screening ( 高通量筛选 )
96-well plate, 384-well plate
HTS uses some well designed models or assays to screen large quantity of compounds in relative short time
In assays, the activities of compounds are visualized: images (in Forward CG) or fluorescent signals (in Reverse CG)
26
Screening Models in Forward Chemical GeneticsScreening Models in Forward Chemical Genetics
Whole Organism Models1. Zebrafish ( 斑马鱼 vertebrate 脊椎动物 )2. Fruit Fly ( 果蝇 )3. C. elegans ( 线虫 )4. Plants ( 植物 )
Cellular Models1. Mammalian Cells ( 哺乳动物细胞 ) 2. Yeast ( 酵母 )
Cell-free SystemRequirements:• Small (96 or 384-well
plate)• Short generation time• Easy to be observed• Inexpensive
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
27
Stem cell differentiation modulatorsStem cell differentiation modulators
purines S1pyrimidines S2quinazolines S3pyrazines S4phthalazines S5pyridazines S6quinoxalines S7
Totally 45140 Compounds
Ding, S., Gray, N.S., Wu, X., Ding, Q. & Schultz, P.G. J. Am. Chem. Soc. 2002. 124, 1594–1596
Kinase Directed Heterocycle Library
28
Stem cell differentiation (Stem cell differentiation ( 干细胞分化干细胞分化 ) modulators) modulators
Ding, S. & Schultz, P.G. Nat. Biotechnol. 2004, 22, 833Tan, D.S. Nature Chem. Biol. 2005, 1(2), 74
a. induces neurogenesis( 神经形成 ) of mouse embryonic stem cellsb. induces cardiomyogenesis( 心形成 ) of mouse embryonic stem cell
sc. induces osteogenesis( 骨生成 ) of mouse mesoderm fibroblast cell
sd. Reversine induces dedifferentiation of myoblasts( 成肌细胞 ) to pro
genitor cells ( 全能细胞 )
Murine stem cell( 鼠的干细胞 ) based phenotypic assays
29
Screening Models in Forward Chemical GeneticsScreening Models in Forward Chemical Genetics
Whole Organism Models1. Zebrafish ( 斑马鱼 vertebrate 脊椎动物 )2. Fruit Fly ( 果蝇 )3. C. elegans ( 线虫 )4. Plants ( 植物 )
Cell Models1. Mammalian Cells ( 哺乳动物细胞 ) 2. Yeast ( 酵母 )
Cell-free SystemRequirements:• Small (96 or 384-well
plate)• Short generation time• Easy to be observed• Inexpensive
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
30
Zebrafish: Development (Zebrafish: Development ( 斑马鱼发育历程斑马鱼发育历程 ))
Pascal Haffter et al. Development, 1996, 123, 1-36
31
Whole Organism Models: ZebrafishWhole Organism Models: Zebrafish
Advantages:• Eggs are small• Short generation time• Cheap • Easy to be observed
(Transparent for the first 5 days)
• Organ systems are very close to their human counterparts
• Good permeability for small molecules
• Large numbers of eggs
Breinbauer, R., Angew. Chem. Int. Ed. 2003, 42, 1086Peterson, R. T., Schreiber, S. L. et al., Proc. Natl. Acad. Sci. 2000, 97, 12965
enlarged hindbrain ventricle ( 后脑室变大 )
folds in the notochord( 脊索弯曲 )
hindbrain abnormality( 后脑异常 )
32
Screening Assays in Reverse Chemical GeneticsScreening Assays in Reverse Chemical Genetics
1. Yeast Three-hybrid System
2. Small Molecular Microarrays
3. Enzyme (Purified Protein Assay)
4. Disruption of Protein-Protein Interactions (Yeast Two-hybrid System )
5. Exploring Receptors and Signal Transduction (Cell Based Assay)
6. ……
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
33
gfp genegfp gene
DBD
LBD
L C PAD
Yeast Three-hybrid SystemYeast Three-hybrid System
Three-Hybrid Components:1. DBD - LBD2. L – C3. P – AD
DBD (DNA bingding dormain) + AD (transcription activation domain)= transcription factor ( 转录激活因子 )
LBD (ligand binding dormain) of a known Ligand
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
Signal amplifierReport gene:
GFP ( 绿色荧光蛋白 )1+2 + 3
L= LignadC= Test Compound (changeable)P= Selected Protein
34
gfp genegfp gene
DBD
LBD
L C PAD
Yeast Three-hybrid SystemYeast Three-hybrid System
Three-Hybrid Components:1. DBD - LBD2. L – C3. P – AD
DBD (DNA bingding dormain) + AD (transcription activation domain)= transcription factor ( 转录激活因子 )
LBD (ligand binding dormain) of a known Ligand
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
Signal amplifierReport gene:
GFP ( 绿色荧光蛋白 )1+2 + 3?
L= LignadC= Test Compound (changeable)P= Selected Protein
35
Purified Protein AssaysPurified Protein Assays
Cheung, A.; Straight, A. F. et al. Nat. Cell Biol. 2002, 4, 83.
Purified myosin subfragment Luciferase( 荧光素酶 )+LuciferinLibrary of 16,300 compounds
N-benzyl-p-toluenesulfonamide (BTS) Myosin inhibitor, IC50= 5uM
Extensively used for the identification of active compounds to enzymes
The activities of molecules are transformed to fluorescent signals which can be read and recorded by machines
GFP( 绿色荧光蛋白 )Luciferase( 荧光素酶 )
36
ContentsContents
Reverse Chemical Genetics and TOS
Forward Chemical Genetics and DOS
High Throughput Screening
Conclusion
37
3. Forward and Reverse Chemical Genetics3. Forward and Reverse Chemical Genetics
ProspectingLibraries
(DOS) Focused Libraries(TOS)
Screening:In vivo
Screening:In vitro
Target identificationPhenotypic response
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
38
Acknowledgement
Prof. Zhen Yang Prof. Zhen Yang Prof. Jiahua ChenProf. Jiahua Chen
Ms. Haixia Zou Ms. Haixia Zou Dr. Zheng Xiang Dr. Zheng Xiang Mr. Qing Xiao Mr. Qing Xiao Mr. Hongbo Yang (Department of Life Science) Mr. Hongbo Yang (Department of Life Science)
All the members in our group All the members in our group
All the members in the Organic InstituteAll the members in the Organic Institute
Target Identification
41
3.1.2 Assays Used in Target Identification3.1.2 Assays Used in Target Identification
Pull Down Assay1. Pull Down Assay
2. Phage Display
Microarrays ( 微阵列 )
1. Protein Microarrays
2. cDNA Microarrays
Yeast Three-hybrid System
Target Identification is Target Identification is the the most difficult partmost difficult part in Chemical Genetics in Chemical Genetics
42
3.1.2.1 Pull Down Assay3.1.2.1 Pull Down Assay
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
West blot(SDS/Page + Silver stain )
GeldanamycinHSP90 inhibitor
Requirements: Attachment to the resin High affinity ligand High abundance of target
43
3.1.3 Case Study3.1.3 Case Study
NH
O
CF3
H3C
N
N O
N
NNH
NN
H3C
SC1
CH3
NH
O
CF3
H3C
N
N O
N
NNH
NN
H3C
O
OO
NH
O
AM-SC1+
Self-renewal of Self-renewal of embryonic stem cellsembryonic stem cells (( 胚胎干细胞胚胎干细胞 )) by a small molecule by a small molecule
Chen S, Ding S et al. Proc Natl Acad Sci USA 2006, 103: 17266-17271
28 out of28 out of5000 compounds5000 compounds
384 well plate384 well plate
44
3.1.2.1.2 Phage Display3.1.2.1.2 Phage Display
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530King R. W., Chem. Biol. 1999, 6, R327-R333
45
3.1.2.2.2 Cellular Microarrays3.1.2.2.2 Cellular Microarrays
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530Zlauddin, J., et al.,Nature 2001, 411, 107-110
46
3.1.2.2 Protein Microarrays3.1.2.2 Protein Microarrays
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
Protein MicroarraysProtein Microarrays
Application: discovery of protein kinase substrates and antigens
Shortcomings:suffer from the lack oflarge numbers of purified and stable proteins available forimmobilization on the microarray surface.
47
gfp genegfp gene
DBD
LBD
L C PAD
3.1.2.3 Yeast Three-hybrid System3.1.2.3 Yeast Three-hybrid System
Three-Hybrid Parts:1. DBD - LBD2. L – C3. P – AD
DBD (DNA bingding dormain) + AD (transcription activation domain)= transcription factor ( 转录激活因子 )
LBD (ligand binding dormain) of a known Ligand
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
Signal amplifierReport gene:
GFP ( 绿色荧光蛋白 )1+2 + 3
L= LignadC= Selected CompoundP= Test Proteins (changeable)