Chemical Genetics

1. 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

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

3. Protein A To improve the activity TOS To improve the selectivity Protein A with Known Inhibitor To design a inhibitor Protein A without Known Inhibitor To Inhibit a Known Protein? Related to Virus(病毒), Cancer or other diseases How to inhibit it?

4. 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.

5. 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

6. TOS Based on Natural Product Epothilones their mechanism of action against tumor cells has been attributed to the binding andstabilization 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 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

8. 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

9. 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

10. 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. 2003:Chapter 7

11. 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

12. TOS Based on Target Protein Structure Active Center HIV Protease (艾滋病毒蛋白酶) Inhibitor designed for HIV protease based on the protein’s crystal structure Hugo Kubinyi. Curr. Opin. in Drug Disc. & Dev. 1998, 41:4

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

14. 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

15. Small Molecules and Targets Proteins Identified in FCG Walsh, D. P., et al., Chem. Rev.2006, 106, 2476-2530

16. Produce eggs Mammalian cells organisms ??? Diverse Library How to Find New Target Protein and New Leading Compound Homology(同源性，相似性) to humans Physiological context 96-well plate, one well one compound

17. 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 • 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.

18. DOS – 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 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.

20. Differentiating Process & Folding Process DOS – Skeletal Diversity Generating 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

21. Differentiating Processes B A C DOS – Skeletal Diversity Generating 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

22. Folding Processes DOS – Skeletal Diversity Generating Process 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

23. 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

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 Genetics • Whole Organism Models • Zebrafish (斑马鱼 vertebrate脊椎动物) • Fruit Fly (果蝇) • C. elegans (线虫) • Plants (植物) • Cellular Models • Mammalian Cells (哺乳动物细胞) • Yeast (酵母) • 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

27. 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

28. Stem cell differentiation (干细胞分化) modulators Murine stem cell(鼠的干细胞) based phenotypic assays • 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

29. Screening Models in Forward Chemical Genetics • Whole Organism Models • Zebrafish (斑马鱼 vertebrate脊椎动物) • Fruit Fly (果蝇) • C. elegans (线虫) • Plants (植物) • Cell Models • Mammalian Cells (哺乳动物细胞) • Yeast (酵母) • 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

30. Zebrafish: Development (斑马鱼发育历程) Pascal Haffter et al. Development, 1996, 123, 1-36

31. Whole Organism Models: Zebrafish enlarged hindbrain ventricle (后脑室变大) 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 (后脑异常) folds in the notochord (脊索弯曲) Breinbauer, R., Angew. Chem. Int. Ed.2003, 42, 1086 Peterson, R. T., Schreiber, S. L. et al., Proc. Natl. Acad. Sci. 2000, 97, 12965

32. 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. P L C AD DBD LBD 1+2 + 3 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 (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. P L C AD DBD LBD 1+2 + 3 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 (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.

35. N-benzyl-p-toluenesulfonamide (BTS) Myosin inhibitor,IC50= 5uM Purified Protein Assays Purified myosin subfragment Luciferase(荧光素酶)+Luciferin 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(绿色荧光蛋白) Luciferase(荧光素酶) Cheung, A.; Straight, A. F. et al. Nat. Cell Biol. 2002, 4, 83.

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

37. Screening: In vitro 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

40. Target Identification

41. 3.1.2 Assays Used in Target Identification • Pull Down Assay • Pull Down Assay • Phage Display • Microarrays (微阵列) • Protein Microarrays • cDNA Microarrays • Yeast Three-hybrid System Target Identification is the most difficult part in Chemical Genetics

42. Geldanamycin HSP90 inhibitor West blot (SDS/Page + Silver stain ) 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(胚胎干细胞) by a small molecule 28 out of 5000 compounds 384 well plate Chen S, Ding S et al. Proc Natl Acad Sci USA2006, 103: 17266-17271

44. 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

45. 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

46. Protein Microarrays 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. P L C AD DBD LBD 1+2 + 3 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 (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.