Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway

1. Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway Ami Jun-Yee Chin February 17, 2005

2. Chemistry Nobel Prize 2004 Awarded to Aaron Cichanover, Avram Hershko, and Irwin Rose .

3. Central Dogma of Molecular Biology DNA TRANSCRIPTION of RNA TRANSLATION to Protein

4. Protein Degradation I - Lysosomal Degradation Lysosome Protein Amino Acids • Activated at times of stress

5. Protein Degradation II – Ubiquitin Proteasome Pathway Proteasome Protein Amino Acids • Housekeeping role • A role in protein regulation

6. Protein Tagging by Ubiquitin • Activation of ubiquitin Ciechanover, A.EMBO J.1998, 17, 7151.

7. Protein Tagging by Ubiquitin • Activation of ubiquitin • Transfer of ubiquitin to a carrier protein

8. Protein Tagging by Ubiquitin • Activation of ubiquitin • Transfer of ubiquitin to a carrier protein • Selection of target protein

9. Formation of Polyubiquitin Chain

10. Protein Recognition • Ubiquitin tag is recognized • Components are recycled and reused

11. Protein Regulation

12. Protein Regulation

13. Protein Regulation

14. Biological Relevance

15. Biological Relevance

16. Lactacystin • Isolated in 1991 • Initially studied as a nerve growth factor • Later found lactacystin to be a proteasome inhibitor Omura, S., et al. J, Antibiot. 1991, 44, 113.

17. Determination of Cellular Target - Lactacystin + Lactacystin Lactacystin was incubated with cell extract Sample was subjected to SDS PAGE Sequencing showed homology to proteasome Schreiber, S.L. et al. Science. 1995, 268, 726.

18. Retrosynthetic Analysis

19. First Total Synthesis Strategy = Self Regeneration of Stereocenters Corey, E.J. and Reichard, G. J. Am. Chem. Soc.1992, 114, 10677.

20. Self Regeneration of Stereocenters Seebach, D. et al. Helv. Chim. Acta. 1987, 70, 1194

21. First Total Synthesis

22. First Total Synthesis Pirrung-Heathcock anti-aldol gave poor diastereoselectivitey

23. Anti-Aldol Closed Transition States

24. First Total Synthesis

25. Drawbacks to Synthesis • Poor diastereoselectivity • Needed to upscale to pursue biological studies

26. Revised Aldol Reaction

27. Revised Aldol Reaction Corey, E.J. et al. J. Am. Chem. Soc. 1998, 120, 2330

28. Magnesium Catalyzed Anti-Aldol Top face is favoured for attack of nucleophile NU NU Bottom face is shielded by Benzyl and OTBS

29. Open Transition State Aldol

30. Improvements to Synthesis • Doubly diastereoselective aldol • Synthesis of lactacystin in kilogram quantities • Quantity allowed further biological investigation

31. SAR Studies of Lactacystin WHAT • Which parts of the target molecule is essential ? HOW • Stepwise changes are made and activity is measured • WHY • To maximize activity of target molecule

32. Initial SAR Studies

33. Initial SAR Studies

34. Mechanistic Studies: In Vitro Dick, L. et al. J. Biol. Chem. 1996, 271, 7273.

35. Mechanistic Studies: In Vitro

36. Mechanistic Studies: In Vitro • Not First order kinetics • Suggests intermediate involved

37. Mechanistic Studies: Hypothesis • Is b-Lactone an intermediate ? • Increasing [NAC]will decrease rate of hydrolysis

38. Effects of [NAC] on Rate of Hydrolysis Addition of NAC impedes rate of hydrolysis

39. HPLC Detection of b-Lactone

40. Mechanistic Studies

41. Mechanistic Studies: Role of Glutathione

42. Mechanistic Studies: Role of Glutathione

43. Mechanistic Studies: Role of Glutathione Can Glutathione react with b-Lactone to give a thioester adduct ? Dick, L. et al. J. Biol. Chem. 1997, 272, 182.

44. Lactathione Formation In Vitro Confirmed Glutathione + b-Lactone Glutathione b-Lactone

45. In Vivo Studies of Lactathione Formation HPLC Cells Washed cells Cell lysate ? HPLC Cells Washed cells Cell lysate

46. In Vivo Studies of Lactathione Formation HPLC Cells Washed cells Cell lysate ? HPLC Cells Washed cells Cell lysate

47. In Vivo Studies of Lactathione Formation HPLC Cells Washed cells Cell lysate ? HPLC Cells Washed cells Cell lysate

48. HPLC Analysis of Cell Extract HPLC Lactacystin HPLC Lactacystin HPLC b-Lactone

49. Fate of Lactacystin In Vivo : 2 Possibilities

50. Fate of Lactacystin In Vivo : 2 Possibilities OR