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 Protein Lysosome Amino Acids  Activated at times of stress

5 Protein Degradation II – Ubiquitin Proteasome Pathway Protein Proteasome 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 was incubated with cell extract Sample was subjected to SDS PAGE Sequencing showed homology to proteasome + Lactacystin - Lactacystin 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,

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 Bottom face is shielded by Benzyl and OTBS NU

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  -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  -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  -Lactone to give a thioester adduct ? Dick, L. et al. J. Biol. Chem. 1997, 272, 182.

44 Lactathione Formation In Vitro Confirmed Glutathione +  -Lactone Glutathione  -Lactone

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

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

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

48 HPLC Analysis of Cell Extract Lactacystin  -Lactone Lactacystin HPLC

49 Fate of Lactacystin In Vivo : 2 Possibilities

50 Fate of Lactacystin In Vivo : 2 Possibilities OR

51 Control with Glutathione Depleted Cells No Glutathione HPLC + Washed cells Cell lysate

52 Control with Glutathione Depleted Cells No Glutathione HPLC + Washed cells Cell lysate

53 Control with Glutathione Depleted Cells No Glutathione HPLC + Washed cells Cell lysate

54 Control with Glutathione Depleted Cells No Glutathione HPLC + Results suggest that Lactacystin Is impermeable to cell membrane Washed cells Cell lysate

55 Mechanism of Action: Role of  -Lactone   -Lactone is the active inhibitor  Only  -Lactone is permeable to cell membrane?

56 Hydrolysis of Lactacystin vs  -Lactone Lactacystin Time (min)  -Lactone]

57  -Lactone] Outside the Cell  - Lactone Lactacystin  Hydrolysis of  -Lactone is slower when starting with Lactacystin Time (min)  -Lactone]

58 Lactathione Accumulation in Cells  Lactathione accumulation is slower in Lactacystin treated cells Time (min) [Lactathione]

59 Mechanism of Action: Conclusions Hydrolysis of  -Lactone  - Lactone Lactacystin Time (min)  -Lactone] Lactathione Accumulation  - Lactone Lactacystin Time (min) [Lactathione] Extracellular [  -Lactone]  Intracellular [Lactathione] CONCLUSION

60 Mechanism of Action: Summary INSIDE CELL OUTSIDE CELL MEMBRANE

61 SAR Studies of Lactacystin  OH and carbonyl are cis   -Lactone formation is necessary for activity

62 SAR Studies of Lactacystin C9 C7

63 Synthesis: C9 Analogues Corey, E. J. et al. Angew. Chem. Int. Ed. 1998, 37, 1676.

64 SAR of C9 Analogues

65 SAR of C7 Analogues

66 SAR Studies of Lactacystin Larger groups Essential Electrophillic Carbonyl Essential

67  -Lactone : Important Feature for Activity  Cell permeability  Electrophillic carbonyl for acylation of proteasome  Isolated by Fenical, 2003  More potent inhibitor than  -Lactone  Cytotoxic activity Fenical, W. et al. Angew. Chem. Int. Ed. 2003, 42, 355.

68 Synthesis of Salinosporamide A Corey, E.J. et al. J. Am. Chem. Soc. 2004, 126, 6230

69 Synthesis of Salinosporamide A

70 Synthesis of Salinosporamide A

71 Synthesis of Salinosporamide A Total synthesis of Salinosporamide A was achieved in 10% over 18 steps

72 Summary: Ubiquitin Proteasome Pathway

73 Summary: Synthesis of Lactacystin

74 Mechanism of Action INSIDE CELL OUTSIDE CELL MEMBRANE

75 Summary: Synthesis of Salinosporamide A Synthesized in 18 steps, 10% overall yield

76 Summary: SAR Studies and Analogs  Can be a larger group  Essential to form  -Lactone  Initially optimized Lactacystin

77 Acknowledgements Dr. Ogilvie Livia Aumand Alison Lemay Mathieu Lemay Matt Clay My Family and Friends And You!