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Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway

Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway. Ami Jun-Yee Chin February 17, 2005. Chemistry Nobel Prize 2004. Awarded to Aaron Cichanover, Avram Hershko, and Irwin Rose. . Central Dogma of Molecular Biology. DNA. TRANSCRIPTION of RNA. TRANSLATION to Protein.

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Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway

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  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

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