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

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

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

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

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

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

Formation of Polyubiquitin Chain

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

Protein Regulation

Biological Relevance

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.

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.

Retrosynthetic Analysis

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

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

First Total Synthesis

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

Anti-Aldol Closed Transition States

First Total Synthesis

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

Revised Aldol Reaction

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

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

Open Transition State Aldol

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

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

Initial SAR Studies

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

Mechanistic Studies: In Vitro

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

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

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

HPLC Detection of b-Lactone

Mechanistic Studies

Mechanistic Studies: Role of Glutathione

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.

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

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

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

Fate of Lactacystin In Vivo : 2 Possibilities

Fate of Lactacystin In Vivo : 2 Possibilities OR

Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway