1. 1 BE 10988 : An Inhibitor of DNA –Topoisomerase II Indira Thapa November 24, 2005

2. 2 DNA DNA Sugar-phosphate backbone Bases H-bonds  Double helical structure  Store genetic information  Uncontrolled cell growth-Cancer

3. 3 DNA - A Molecular Target for Cancer Therapeutics  DNA-DNA crosslinker e.g. mitomycin C  Intercalator e.g. ellipticine  Double-stranded break e.g. bleomycin Develop drug resistance and mutagenesis  DNA binding during replication - Topoisomerase II New anticancer drug target Protein-DNA complex

4. 4 Overview  DNA Replication and Supercoiling  Topoisomerase II - A nuclear enzyme -Importance and functions in cellular processes -Enzymatic action mechanism & interaction with inhibitor  BE 10988 -Biological properties -Total synthesis  Structure Activity Relationship (SAR) Studies  Summary

5. 5 DNA Replication  DNA helix rotation during replication

6. 6 Supercoil formed during replication DNA Supercoiling  Supercoiling - coiling of coils

7. 7 Topoisomerase II (Topo II ) FUNCTIONS: transiently breaks and reseals double stranded DNA simultaneously, and allows the passage of separate double helical strand through the break site.  Eukaryotic topoisomerases - type I and type II  Homologous dimer  Separate intact DNA

8. 8 Mechanism of Action of Topoisomerase II “Cleavable complex” N-terminal C-terminal

9. 9 Action of Inhibitor Inhibitor stabilizes “Cleavable complex” Permanent DNA double strand break Cell Death Blocks re-ligation inhibitor

10. 10 Interest in Topo II Inhibitors Interest in Topo II Inhibitors  Without Topo II DNA cannot replicate normally  Inhibitors of topoisomerase II have been used as anticancer drugs  Proliferating tumour cells show high levels of topo II activity and this proliferative activity has in turn been associated with increased tumour cell sensitivity to topoisomerase II – interactive drugs.  Selective inhibition of topoisomerase II in cancer cells could lead to a new approach to cancer therapy

11. 11 BE 10988  Isolated from the culture broth of a strain of actinomycetes in 1991  Potent topoisomerase II inhibitor Shizuri, Y. et. al. J. Antibiot. 1991, 44, 486-491

12. 12 Biological Properties Shizuri, Y. et. al. J. Antibiot. 1991, 44, 486-491 Relaxed Plasmid DNA  Showed growth inhibitory activity against resistant mouse tumour cells  Topoisomerase II-DNA complex formed  Inhibited the relaxation of plasmid DNA Supercoiled Plasmid DNA

13. 13 First Total Synthesis-Retrosynthesis Moody, C. J.; Jones, G. B.; J. Chem. Soc. Perkin Trans. 1, 1989, 2455 Moody, C. J.; Swann, E.; J. Chem. Soc. Perkin Trans. 1, 1993, 2561

14. 14 First Total Synthesis Moody, C. J.; Jones, G. B.; J. Chem. Soc. Perkin Trans. 1, 1989, 2455 Moody, C.J.; Swann, E.; J. Chem. Soc. Perkin Trans. 1, 1993, 2561

15. 15 First Total Synthesis

16. 16 First Total Synthesis

17. 17 First Total Synthesis

18. 18 First Total Synthesis

19. 19 First Total Synthesis BE 10988

20. 20 Nicolaou, K. C. et al. J. Am. Chem. Soc. 2002, 124, 2221-2232 Synthesis Nicolaou’s Synthesis Retrosynthetic - Analysis

21. 21 Nicolaou’s Synthesis Nicolaou, K. C. et al. J. Am. Chem. Soc. 2002, 124, 2221-2232

22. 22 Nicolaou’s Synthesis

23. 23 Proposed Mechanism

24. 24 p-quinone Formation

25. 25 H 2 O 18 Isotope Labeling Studies

26. 26 BE 10988 – Nicolaou’s Synthesis BE 10988

27. 27 Summary : Total Synthesis 18 steps 28% overall yield Moody’s synthesis: 1993 Nicolaou’s synthesis: 2002 24% overall yield 5 steps

28. 28 SAR Studies SAR Studies Quinone system Thiazole moeity Which part of the molecule is essential ? Bailly. C. et. al. Bioorg. Med. Chem. Lett. 9, 1999 2025-2030

29. 29 Analogues of BE 10988 1 2 3 4 Bailly. C. et. al. Bioorg. Med. Chem. Lett. 9, 1999, 2025-2030

30. 30 Growth Growth Inhibitory Activity Mouse leukemia cells Mouse leukemia cells + + IC 50 (µM) > 10 0.8 Indolequinone skeleton is essential to the activity

31. 31 Growth Inhibitory Activity + + Mouse Leukemia cells Mouse leukemia cells IC 50 (µM) > 100 6.5 Thiazole ring is also important for the activity  Analogue 4 is 8 fold less active than analogue 2

32. 32 Reaction Catalyzed By Topo II Decatenation Catenated circular DNA Decatenated circular DNA  prokaryotic system  simple plasmid DNA

33. 33 Topo II Inhibition: Decatenation Assay KDNA (catenated circular DNA) thiazolylindolequinone Morpholino derivative SDS Proteinase K KDNA SDS Proteinase K Standard for comparision Electrophoresis Etoposide Topoisomerase II

34. 34 Topo II inhibition: Decatenation Assay 50µM Effective

35. 35 Linear DNA Circular plasmid DNA DNA Cleavage Assay Drug inhibits re-ligation of DNA once the double helix is cleaved by the enzyme

36. 36 Summary : SAR Studies Quinone system is essential to activity Thiazole moeity plays a major role in topoisomerase II inhibition  Analogue 2 is weak inhibitor of topoisomerase II, suggests that - NH 2 substituent at quinone ring is also important for activity Required

37. 37 DNA Binding  Non-specific binding  Analogues do not intercalate into DNA  Study supports the fact that BE 10988 does not bind DNA covalently Further studies have Shown:

38. 38 Conclusions  Two total syntheses of BE 10988  SAR studies of BE 10988 using four different analogues  More detailed mechanistic studies need to be done on BE 10988

39. 39 Acknowledgements Dr. Robert Ben Ben Research Group Vincent Bouvet Frank Cease Suhuai Liu Pawel Czechura Elisabeth von Moos Roger Tam Jennifer Chaytor Jessica Jackman Nicole Le Grand Aleksandra Paliga Alison Lemay and …You

40. 40 Mechanistic Studies  The new oxygen atom in the reaction is derived from Ac-IBX.

41. 41 Compds 2 & 4 in presence of DNA DNA Binding  Non –specific binding of drugs to DNA  Drugs trigger double stranded DNA cleavage via topoisomerase II Compds 2 & 4

42. 42 inhibitor Formation of Ternary Complex inhibitor Route 2 Route 1Route 3 Fortune JM, et. al,; Prog Nucleic acid Res Mol Bio 64:221-253

43. 43 Decarbonylation