Chemical Genetics

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Chemical Genetics
OPSS
--------Exploring the intersections
between chemistry and biology
Supervisors Prof. Zhen Yang
Prof. Jiahua Chen Report Jing
Xiang(??) 2007-6-8
2
Contents

• Reverse Chemical Genetics and TOS
• Forward Chemical Genetics and DOS
• High Throughput Screening
• Conclusion

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To Inhibit a Known Protein?
Related to Virus(??), Cancer or other diseases
How to inhibit it?
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TOS (Target Oriented Synthesis)
To synthesize a collection of compounds based on
a target
Focused Library (librarya 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.
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TOS Based on Natural Products

• 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
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TOS Based on Natural Product
Epothilones
their mechanism of action against tumor cells has
been attributed to the binding and stabilization
of microtubules(??,?????????).
Nicolaou, K. C. et al. Angew. Chem. Int. Ed.
Engl. 1998, 37, 2014
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TOS 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
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TOS 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
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TOS Based Natural Product
Building Blocks
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
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TOS Based on Nature Products
Better Activity
Structure Activity Relationship
Nicolaou, K. C. and Snyder, S. A.. Classics in
Total Synthesis II More Target, Strategies,
Methods. WILEY-VCH GmbH Co. KGaA. 2003Chapter
7
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TOS Based on Drug Candidates
Better Selectivity
1 VEGFR2/3 inhibitor Not specific
Specific inhibitor VEGFR2 IC500.25nm
Christian Peifer et al. J. Med. Chem. 2006, 49,
1271-1281
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TOS Based on Target Protein Structure
Active Center
HIV Protease (???????)
Inhibitor designed for HIV protease based on the
proteins crystal structure
Hugo Kubinyi. Curr. Opin. in Drug Disc. Dev.
1998, 414
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Contents

• Reverse Chemical Genetics and TOS
• Forward Chemical Genetics and DOS
• High Throughput Screening
• Conclusion

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Human Genome (?????)

• 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
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Small Molecules and Targets Proteins Identified
in FCG
Walsh, D. P., et al., Chem. Rev. 2006, 106,
2476-2530
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How to Find New Target Protein and New Leading
Compound
Homology(???,???) to humans
Physiological context
96-well plate, one well one compound
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Resource of Diverse Library
Commercial Libraries
Available gt1500 compound libraries (data of
2005) Not Available Pharmaceutical companies
DOS (Diversity Oriented Synthesis)

• Prospecting Library
• Solid Phase Synthesis
• Structure Diverse Molecules
• Diversity Generating Processes
• Appendage Diversity Benzopyrans, Shikimic Acid
• Stereochemical Diversity 32, 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.
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DOS Appendage Diversity Generating Process
Nicolaou, K. C. et al. J. Am. Chem. Soc. 2000,
122, 9939-53, 9954-67, 9968-76.
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DOS Stereochemical Diversity Generating Process
Catalytic Asymmetric 32 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.
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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
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DOS Skeletal Diversity Generating Processes

• Differentiating Processes

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
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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
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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
24
Contents

• Reverse Chemical Genetics and TOS
• Forward Chemical Genetics and DOS
• High Throughput Screening
• Conclusion

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High Throughput Screening (?????)
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)
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Screening Models in Forward Chemical Genetics

• Whole Organism Models
• Zebrafish (??? vertebrate????)
• Fruit Fly (??)
• C. elegans (??)
• Plants (??)
• Cellular Models
• Mammalian Cells (??????)
• Yeast (??)
• Cell-free System

• Requirements
• 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
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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, 15941596
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Stem cell differentiation (?????) modulators
Murine stem cell(?????) based phenotypic assays

1. induces neurogenesis(????) of mouse embryonic
   stem cells
2. induces cardiomyogenesis(???) of mouse embryonic
   stem cells
3. induces osteogenesis(???) of mouse mesoderm
   fibroblast cells
4. Reversine induces dedifferentiation of
   myoblasts(????) to progenitor cells (????)

Ding, S. Schultz, P.G. Nat. Biotechnol. 2004,
22, 833 Tan, D.S. Nature Chem. Biol. 2005, 1(2),
74
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Screening Models in Forward Chemical Genetics

• Whole Organism Models
• Zebrafish (??? vertebrate????)
• Fruit Fly (??)
• C. elegans (??)
• Plants (??)
• Cell Models
• Mammalian Cells (??????)
• Yeast (??)
• Cell-free System

• Requirements
• 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
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Zebrafish Development (???????)
Pascal Haffter et al. Development, 1996, 123,
1-36
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Whole Organism Models Zebrafish
enlarged hindbrain ventricle (?????)

• 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

hindbrain abnormality (????)
folds in the notochord (????)
Breinbauer, R., Angew. Chem. Int. Ed. 2003, 42,
1086 Peterson, R. T., Schreiber, S. L. et al.,
Proc. Natl. Acad. Sci. 2000, 97, 12965
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Screening Assays in Reverse Chemical Genetics

• Yeast Three-hybrid System
• Small Molecular Microarrays
• Enzyme (Purified Protein Assay)
• Disruption of Protein-Protein Interactions
  (Yeast Two-hybrid System )
• Exploring Receptors and Signal Transduction
  (Cell Based Assay)

Walsh, D. P., et al., Chem. Rev. 2006, 106,
2476-2530
33
Yeast Three-hybrid System
L Lignad C Test Compound (changeable) P
Selected Protein
gfp gene
Three-Hybrid Components 1. DBD - LBD 2. L C 3.
P AD
Signal amplifier Report gene GFP (??????)
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) AD (transcription
activation domain) transcription factor (??????)
Licitra, E. J. Liu, J. O. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 12817.
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Yeast Three-hybrid System
L Lignad C Test Compound (changeable) P
Selected Protein
gfp gene
Three-Hybrid Components 1. DBD - LBD 2. L C 3.
P AD
Signal amplifier Report gene GFP (??????)
?
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) AD (transcription
activation domain) transcription factor (??????)
Licitra, E. J. Liu, J. O. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 12817.
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Purified Protein Assays
Purified myosin subfragment Luciferase(????)Luci
ferin Library of 16,300 compounds
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(??????) Luciferase(????)
Cheung, A. Straight, A. F. et al. Nat. Cell
Biol. 2002, 4, 83.
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Contents

• Reverse Chemical Genetics and TOS
• Forward Chemical Genetics and DOS
• High Throughput Screening
• Conclusion

37
3. Forward and Reverse Chemical Genetics
Prospecting Libraries (DOS)
Focused Libraries (TOS)
Screening In vivo
Phenotypic response
Target identification
Walsh, D. P., et al., Chem. Rev. 2006, 106, 2476
38
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

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Target Identification
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3.1.2 Assays Used in Target Identification

• Pull Down Assay
• Pull Down Assay
• Phage Display
• Microarrays (???)
• Protein Microarrays
• cDNA Microarrays
• Yeast Three-hybrid System

Target Identification is the most difficult part
in Chemical Genetics
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3.1.2.1 Pull Down Assay
Requirements Attachment to the resin
High affinity ligand
High abundance of target
Walsh, D. P., et al., Chem. Rev. 2006, 106,
2476-2530
43
3.1.3 Case Study
Self-renewal of embryonic stem cells (?????) by a
small molecule
28 out of 5000 compounds 384 well plate
Chen S, Ding S et al. Proc Natl Acad Sci USA
2006, 103 17266-17271
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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
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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
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3.1.2.2 Protein Microarrays
Application discovery of protein kinase
substrates and antigens Shortcomings 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
47
3.1.2.3 Yeast Three-hybrid System
L Lignad C Selected Compound P Test Proteins
(changeable)
gfp gene
Three-Hybrid Parts 1. DBD - LBD 2. L C 3. P
AD
Signal amplifier Report gene GFP (??????)
LBD (ligand binding dormain) of a known Ligand
DBD (DNA bingding dormain) AD (transcription
activation domain) transcription factor (??????)
Licitra, E. J. Liu, J. O. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 12817.