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

High Throughput Screening

Conclusion
2
To Inhibit a Known Protein?
Related to Virus(病毒), Cancer or other diseases
Protein A
How to inhibit it?
To improve the activity
Protein A with
Known Inhibitor
To improve the selectivity
TOS
To design a inhibitor
Protein A without
Known Inhibitor
3
TOS (Target Oriented Synthesis)
To synthesize a collection of compounds based on a target
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.
4
TOS Based on Natural Products
1.
2.
3.
diversity
new structure
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
5
TOS 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
6
TOS Based on Natural Product
The Olefin Metathesis Approach of Epothilone A
OHC
A
LDA. THF
HO
COOH
COOH
O
OTBS
O
OTBS
4
B
S
DCC, 4-DMAP
N
C
OH
PCy3
Cl
Ru
S
Cl
HO
N
Ph
PCy3
S
HO
N
50%
O
OTBS
(45% overall yield
from B)
O
O
6
O
O
OTBS
5
1. CF3COOH (20 voI.%), CH2CI2
(98%)
2. mCPBA, benzene
(55%)
O
S
HO
N
O
O
OH
O
epothilone A
Yang, Z.; He, Y.; Vourloumis, D.; Vallberg, H.; Nicolaou, K.C. Angew. Chem. Int. Ed. Engl. 1997, 36, 166
7
TOS Based on Natural Product
Retro-synthetic Analysis of TOS
Metathesis
Epoxidation
O
Me
HO
S
Me
S
Me
Me
HO
N
N
O
Me
O
OH
O
Me
O
O
OR
1
O
2
O
H
O
4
O
Me
OH
HO
O
OR
Me
O
5
S
Me
N
OH
Me
Aldol
condensation
S
Me
N
O
O
OR
O
Esterification
3
6
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
8
TOS Based Natural Product
Building Blocks
O
H
O
O
H
R
O
H
O
R2
O
H
O
OH
O
O
O
OH
O
OH
OTBSO
O
S
OTBSO
OTBSO
S
Me
N
R3
O
S
Me
Me
OH
Ph
N
OH
N
OH
OH
S
Me
OH
N
OH
N
OH
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
9
TOS Based on Nature Products
Structure Activity Relationship
Better Activity
Nicolaou, K. C. and Snyder, S. A.. Classics in Total Synthesis II: More Target, Strategies, Methods.
WILEY-VCH GmbH & Co. KGaA. 2003:Chapter 7
10
TOS Based on Drug Candidates
Better Selectivity
1
VEGFR2/3 inhibitor
Not specific
Specific inhibitor
VEGFR2
IC50=0.25nm
Christian Peifer et al. J. Med. Chem. 2006, 49, 1271-1281
11
TOS Based on Target Protein Structure
Active Center
Gly 48
O
Gly 48'
N
H
O
HO
Ile 50
Ile 50'
N
H
N
H
NH
HN
HN
O
N
H
HIV Protease
(艾滋病毒蛋白酶)
OH
NH
O
O
O
H
N
N
O
Asp 30
N
O
OH
O
O
OH
Asp 25
O
H
N
O
Asp 25'
Asp 30'
Inhibitor
designed
for HIV protease
based on the
protein’s crystal
structure
Hugo Kubinyi. Curr. Opin. in Drug Disc. & Dev. 1998, 41:4
12
Contents

Reverse Chemical Genetics and TOS

Forward Chemical Genetics and DOS

High Throughput Screening

Conclusion
13
Human Genome (人类基因组)
~30,000 Genes
~100,000 Proteins
 fewer than 500 proteins
have fairly well-elucidated
biological functions
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
J. Drews, Nature Biotech. 1996, 30, 97-108
A.L. Hopkins, C.R. Groom, Nat. Rev. Drug Discov. 2002, 1, 727-730
14
Small Molecules and Targets Proteins Identified in FCG
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
15
How to Find New Target Protein and New Leading Compound
Produce
eggs
Mammalian cells
Homology(同源性，相似性) to humans
organisms
Physiological context
Diverse Library
???
96-well plate, one well one compound
16
Resource of Diverse Library
Commercial Libraries
Available: >1500 compound libraries (data of 2005)
Not Available: Pharmaceutical companies
DOS (Diversity Oriented Synthesis)
Prospecting Library
Solid Phase Synthesis
Structure Diverse Molecules:
Diversity Generating Processes
1.
2.
3.
Appendage Diversity: Benzopyrans, Shikimic Acid
Stereochemical Diversity: [3+2], D-A
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.
17
DOS – Appendage Diversity Generating Process
Nicolaou, K. C. et al. J. Am. Chem. Soc. 2000, 122, 9939-53, 9954-67, 9968-76.
18
DOS – Stereochemical Diversity Generating Process
Catalytic Asymmetric [3+2] Cycloaddition of Azomethine Ylides.
Stereochemical diversification of up to 4 tetrahedral centers
Chen, C.; Li, X.; Schreiber, S. L. J. Am. Chem. Soc. 2003, 125, 10174.
19
DOS – Skeletal Diversity Generating Processes
Differentiating Process &
Folding Process
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46-58
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
20
DOS – Skeletal Diversity Generating Processes
Differentiating Processes
B
A
C
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46-58
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
21
DOS – Skeletal Diversity Generating Process
Folding Processes
cis-enedione intermediate
Burke, M.D., Schreiber, S.L., Angew. Chem. Int. Ed. 2004, 43, 46
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
22
Forward and Reverse Chemical Genetics
How to determine the effect of the compounds in Libraries?
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
23
Contents

Reverse Chemical Genetics and TOS

Forward Chemical Genetics and DOS

High Throughput Screening

Conclusion
24
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)
25
Screening Models in Forward Chemical Genetics

Whole Organism Models
1.
4.
Zebrafish (斑马鱼 vertebrate脊椎动物)
Fruit Fly (果蝇)
C. elegans (线虫)
Plants (植物)

Cellular Models
1.
Mammalian Cells (哺乳动物细胞)
Yeast (酵母)
2.
3.
2.
Requirements:

Cell-free System
•
•
•
•
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
26
Stem cell differentiation modulators
Kinase Directed Heterocycle
Library
purines S1
pyrimidines S2
quinazolines S3
pyrazines S4
phthalazines S5
pyridazines S6
quinoxalines S7
Totally 45140 Compounds
Ding, S., Gray, N.S., Wu, X., Ding, Q. & Schultz, P.G.
J. Am. Chem. Soc. 2002. 124, 1594–1596
27
Stem cell differentiation (干细胞分化) modulators
Murine stem cell(鼠的干细胞) based phenotypic assays
a.
b.
c.
d.
induces neurogenesis(神经形成) of mouse embryonic stem cells
induces cardiomyogenesis(心形成) of mouse embryonic stem cells
induces osteogenesis(骨生成) of mouse mesoderm fibroblast cells
Reversine induces dedifferentiation of myoblasts(成肌细胞) to
progenitor cells (全能细胞)
Ding, S. & Schultz, P.G. Nat. Biotechnol. 2004, 22, 833
Tan, D.S. Nature Chem. Biol. 2005, 1(2), 74
28
Screening Models in Forward Chemical Genetics

Whole Organism Models
1.
4.
Zebrafish (斑马鱼 vertebrate脊椎动物)
Fruit Fly (果蝇)
C. elegans (线虫)
Plants (植物)

Cell Models
1.
Mammalian Cells (哺乳动物细胞)
Yeast (酵母)
2.
3.
2.
Requirements:

Cell-free System
•
•
•
•
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
29
Zebrafish: Development (斑马鱼发育历程)
Pascal Haffter et al. Development, 1996, 123, 1-36
30
Whole Organism Models: Zebrafish
enlarged hindbrain ventricle
(后脑室变大)
Advantages:
•
•
•
•
•
•
•
folds in the notochord
(脊索弯曲)
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
hindbrain abnormality
(后脑异常)
Breinbauer, R., Angew. Chem. Int. Ed. 2003, 42, 1086
Peterson, R. T., Schreiber, S. L. et al., Proc. Natl. Acad. Sci. 2000, 97, 12965
31
Screening 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
32
Yeast Three-hybrid System
L
C
P
AD
LBD
DBD
L= Lignad
C= Test Compound
(changeable)
P= Selected Protein
gfp gene
Three-Hybrid Components:
1. DBD - LBD
2. L – C
3. P – AD
1+2 + 3
Signal amplifier
Report gene:
GFP (绿色荧光蛋白)
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) + AD (transcription activation domain)
= transcription factor (转录激活因子)
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
33
Yeast Three-hybrid System
L
C
P
AD
LBD
DBD
L= Lignad
C= Test Compound
(changeable)
P= Selected Protein
gfp gene
Three-Hybrid Components:
1. DBD - LBD
2. L – C
3. P – AD
?
1+2 + 3
Signal amplifier
Report gene:
GFP (绿色荧光蛋白)
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) + AD (transcription activation domain)
= transcription factor (转录激活因子)
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
34
Purified Protein Assays
Extensively used for the
identification of active
compounds to enzymes
Purified myosin subfragment
Luciferase(荧光素酶)+Luciferin
Library of 16,300 compounds
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)
N-benzyl-p-toluenesulfonamide (BTS)
Myosin inhibitor, IC50= 5uM
Cheung, A.; Straight, A. F. et al. Nat. Cell Biol. 2002, 4, 83.
35
Contents

Reverse Chemical Genetics and TOS

Forward Chemical Genetics and DOS

High Throughput Screening

Conclusion
36
3. Forward and Reverse Chemical Genetics
Prospecting
Libraries
(DOS)
Focused
Libraries
(TOS)
Screening:
In vitro
Screening:
In vivo
Target identification
Phenotypic response
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
37
Acknowledgement

Prof. Zhen Yang
Prof. Jiahua Chen

Ms. Haixia Zou
Dr. Zheng Xiang
Mr. Qing Xiao
Mr. Hongbo Yang (Department of Life Science)

All the members in our group

All the members in the Organic Institute
38
Target Identification
3.1.2 Assays Used in Target Identification

Pull Down Assay
1.
2.
Pull Down Assay
Phage Display

Microarrays (微阵列)
1.
2.
Protein Microarrays
cDNA Microarrays

Yeast Three-hybrid System
Target Identification is
the most difficult part in Chemical Genetics
41
3.1.2.1 Pull Down Assay
Geldanamycin
HSP90 inhibitor
West blot
(SDS/Page + Silver stain )
Requirements: Attachment to the resin
High affinity ligand
High abundance of target
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
42
3.1.3 Case Study
Self-renewal of embryonic stem cells (胚胎干细胞) by a small molecule
28 out of
5000 compounds
384 well plate
H3C
O
H3C
N
N
H3C
H3C
N
N
N
N
H
CH3
N
N
N
N
H
N
O
N
H
O
SC1
CF3
N
N
N
N
H
CF3
O
O
O
O
O
N
H
AM-SC1+
Chen S, Ding S et al. Proc Natl Acad Sci USA 2006, 103: 17266-17271
43
3.1.2.1.2 Phage Display
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
King R. W., Chem. Biol. 1999, 6, R327-R333
44
3.1.2.2.2 Cellular Microarrays
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
Zlauddin, J., et al.,Nature 2001, 411, 107-110
45
3.1.2.2 Protein Microarrays
Application:
discovery of protein
kinase substrates and
antigens
Shortcomings:
Protein Microarrays
suffer from the lack of
large numbers of purified
and stable proteins
available for
immobilization on the
microarray surface.
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476-2530
46
3.1.2.3 Yeast Three-hybrid System
L
C
P
AD
LBD
DBD
L= Lignad
C= Selected Compound
P= Test Proteins
(changeable)
gfp gene
Three-Hybrid Parts:
1. DBD - LBD
2. L – C
3. P – AD
1+2 + 3
Signal amplifier
Report gene:
GFP (绿色荧光蛋白)
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) + AD (transcription activation domain)
= transcription factor (转录激活因子)
Licitra, E. J.; Liu, J. O. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817.
47