Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway

1
Lactacystin An Inhibitor in the Ubiquitin
Proteasome Pathway

• Ami Jun-Yee Chin
• February 17, 2005

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Chemistry Nobel Prize 2004

• Awarded to Aaron Cichanover,
• Avram Hershko, and Irwin Rose

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

• I - Lysosomal Degradation

Lysosome
Protein
Amino Acids

• Activated at times of stress

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Protein Degradation

• II Ubiquitin Proteasome Pathway

Proteasome
Protein
Amino Acids

• Housekeeping role
• A role in protein regulation

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Protein Tagging by Ubiquitin

• Activation of ubiquitin

Ciechanover, A. EMBO J. 1998, 17, 7151.
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Protein Tagging by Ubiquitin

• Activation of ubiquitin

• Transfer of ubiquitin to a carrier protein

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Protein Tagging by Ubiquitin

• Activation of ubiquitin

• Transfer of ubiquitin to a carrier protein

• Selection of target protein

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Formation of Polyubiquitin Chain
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Protein Recognition

• Ubiquitin tag is recognized
• Components are recycled and reused

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Protein Regulation
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Protein Regulation
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Protein Regulation
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Biological Relevance
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Biological Relevance
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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.
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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.
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Retrosynthetic Analysis
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First Total Synthesis
Strategy Self Regeneration
of Stereocenters
Corey, E.J. and Reichard, G. J. Am. Chem. Soc.
1992, 114, 10677.
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Self Regeneration of Stereocenters
Seebach, D. et al. Helv. Chim. Acta. 1987, 70,
1194
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First Total Synthesis
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First Total Synthesis
Pirrung-Heathcock anti-aldol gave
poor diastereoselectivitey
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Anti-Aldol Closed Transition States
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First Total Synthesis
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Drawbacks to Synthesis

• Poor diastereoselectivity
• Needed to upscale to pursue biological studies

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Revised Aldol Reaction
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Revised Aldol Reaction
Corey, E.J. et al. J. Am. Chem. Soc. 1998, 120,
2330
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Magnesium Catalyzed Anti-Aldol
Top face is favoured for attack of nucleophile
NU
NU
Bottom face is shielded by Benzyl and OTBS
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Open Transition State Aldol
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Improvements to Synthesis

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

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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

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

• Not First order kinetics
• Suggests intermediate
• involved

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Mechanistic Studies Hypothesis

• Is b-Lactone an intermediate ?
• Increasing NAC will decrease rate of hydrolysis

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Effects of NAC on Rate of Hydrolysis
Addition of NAC impedes rate of hydrolysis
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HPLC Detection of b-Lactone
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Mechanistic Studies
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Mechanistic Studies Role of Glutathione
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Mechanistic Studies Role of Glutathione
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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.
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Lactathione Formation In Vitro Confirmed
Glutathione b-Lactone
Glutathione
b-Lactone
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In Vivo Studies of Lactathione Formation
HPLC
Cells
Washed cells
Cell lysate
?
HPLC
Cells
Washed cells
Cell lysate
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In Vivo Studies of Lactathione Formation
HPLC
Cells
Washed cells
Cell lysate
?
HPLC
Cells
Washed cells
Cell lysate
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In Vivo Studies of Lactathione Formation
HPLC
Cells
Washed cells
Cell lysate
?
HPLC
Cells
Washed cells
Cell lysate
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HPLC Analysis of Cell Extract
HPLC
Lactacystin
HPLC
Lactacystin
HPLC
b-Lactone
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Fate of Lactacystin In Vivo 2 Possibilities
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Fate of Lactacystin In Vivo 2 Possibilities
OR
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Control with Glutathione Depleted Cells
Cell lysate
HPLC

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

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

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

Washed cells
No Glutathione
Results suggest that Lactacystin Is impermeable
to cell membrane
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Mechanism of Action Role of b-Lactone

• b-Lactone is the active inhibitor
• Only b-Lactone is permeable to cell membrane?

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Hydrolysis of Lactacystin vs b-Lactone
b-Lactone
Lactacystin
Time (min)
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b-Lactone Outside the Cell
b- Lactone

• Hydrolysis of b-Lactone
• is slower when starting
• with Lactacystin

b-Lactone
Lactacystin
Time (min)
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Lactathione Accumulation in Cells

• Lactathione accumulation is slower in Lactacystin
  treated cells

Lactathione
Time (min)
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Mechanism of Action Conclusions
b- Lactone
b- Lactone
Lactacystin
b-Lactone
Lactacystin
Lactathione
Time (min)
Time (min)
Hydrolysis of b-Lactone
Lactathione Accumulation
CONCLUSION
Extracellular b-Lactone a Intracellular
Lactathione
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Mechanism of Action Summary
INSIDE CELL
MEMBRANE
OUTSIDE CELL
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SAR Studies of Lactacystin

• OH and carbonyl are cis
• b-Lactone formation is necessary for activity

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SAR Studies of Lactacystin
C7
C9
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Synthesis C9 Analogues
Corey, E. J. et al. Angew. Chem. Int. Ed. 1998,
37, 1676.
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SAR of C9 Analogues
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SAR of C7 Analogues
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SAR Studies of Lactacystin
Larger groups
Essential
Electrophillic Carbonyl
Essential
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b-Lactone Important Feature for Activity

• Cell permeability
• Electrophillic carbonyl for
• acylation of proteasome

• Isolated by Fenical, 2003
• More potent inhibitor
• than b-Lactone
• Cytotoxic activity

Fenical, W. et al. Angew. Chem. Int. Ed. 2003,
42, 355.
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Synthesis of Salinosporamide A
Corey, E.J. et al. J. Am. Chem. Soc. 2004, 126,
6230
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Synthesis of Salinosporamide A
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Synthesis of Salinosporamide A
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Synthesis of Salinosporamide A
Total synthesis of Salinosporamide A was
achieved in 10 over 18 steps
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Summary Ubiquitin Proteasome Pathway
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Summary Synthesis of Lactacystin
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Mechanism of Action
INSIDE CELL
MEMBRANE
OUTSIDE CELL
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Summary Synthesis of Salinosporamide A
Synthesized in 18 steps, 10 overall yield
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Summary SAR Studies and Analogs
Lactacystin

• Can be a larger group
• Essential to form b-Lactone
• Initially optimized

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