BE 10988 : An Inhibitor of DNA –Topoisomerase II

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

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

3. DNA - A Molecular Target for Cancer Therapeutics Protein-DNA complex New anticancer drug target • 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

4. 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 Overview

5. DNA Replication • DNA helix rotation during replication

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

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

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

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

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

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. 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. First Total Synthesis

16. First Total Synthesis

17. First Total Synthesis

18. First Total Synthesis

19. First Total Synthesis BE 10988

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

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

22. Nicolaou’s Synthesis

23. Proposed Mechanism

24. p-quinone Formation

25. H2O18 Isotope Labeling Studies

26. BE 10988 – Nicolaou’s Synthesis BE 10988

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

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

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

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

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

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

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

34. Topo II inhibition: Decatenation Assay Effective 50µM

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

36. Summary : SAR Studies Thiazole moeity plays a major role in topoisomerase II inhibition Required Quinone system is essential to activity • Analogue 2 is weak inhibitor of topoisomerase II, suggests that - NH2 substituent at quinone ring is also important for activity

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

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

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

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

43. Decarbonylation