Chemical Genetics

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

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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

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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)