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Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway Ami Jun-Yee Chin February 17, 2005
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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
RNA
PROTEIN
DNA
TRANSCRIPTION of RNA
TRANSLATION to Protein
4
Protein Degradation
I - Lysosomal Degradation
ProteinLysosome
Amino Acids
Activated at times of stress
5
Protein Degradation
II – Ubiquitin Proteasome Pathway
ProteinProteasome
Amino Acids
Housekeeping role A role in protein regulation
6
Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SH
OS E2
E2 SH
ON Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Ciechanover, A. EMBO J. 1998, 17, 7151.
7
Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SH
OS E2
E2 SH
ON Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Transfer of ubiquitin to a carrier protein
8
Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SH
OS E2
E2 SH
ON Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Transfer of ubiquitin to a carrier protein
Selection of target protein
9
Formation of Polyubiquitin Chain
UBIQUITIN
TARGET PROTEIN
E3
E2 SH
E1 SH
O
NH
Lys
O
NH
Lys
O
N Lys
O
NH
Lys
1.
2.
3.
10
Protein Recognition
UBIQUITIN
TARGET PROTEIN
Ubiquitin tag is recognized
Components are recycled and reused
O
NH
Lys
O
NH
Lys
O
NH
Lys
Amino Acids Recycled Ubiquitin
O
NH
Lys
O
NH
Lys
O
NH
Lys
11
Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
Ubiquitin Tag
12
Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
13
Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
14
Biological Relevance
TF-
INFLAMMATORY PROTEINS
I-
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
15
Biological Relevance
INHIBITEDPROTEASOME
I-
TF-
INFLAMMATORY PROTEINS
NO
NUCLEUS
16
Lactacystin
NH
O
O
SNHAc
HO2C
Me
HOHO
(+) - 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
NH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin wasincubated withcell extract
Sample wassubjected to SDS PAGE
Sequencing showed homologyto proteasome
+ Lactacystin- Lactacystin
Schreiber, S.L. et al. Science. 1995, 268, 726.
18
Retrosynthetic Analysis
O
N
Ph
OTBS
OHO
Me
ONH
OMe
HO O
SNHAc
HO2C
HO
H2N
HO
CO2Me
O
N
PhCO2Me
MeO2C
HNOH
Ph
OH
19
First Total Synthesis NH
O
O
SNHAc
HO2C
Me
HOHO
O
N
PhCO2Me
H
O
O
N
Ph
HO
CO2Me
PhCH2Br
O
HN CO2Me
MeO2C
HNOH
Ph
OH
O
HH2N
HO
CO2Me
LDA, LiBr
MeOH, TfOH
91%
84% 52%
51%
Strategy = Self Regeneration of Stereocenters
Corey, E.J. and Reichard, G. J. Am. Chem. Soc. 1992, 114, 10677.
20
Self Regeneration of Stereocenters
HN
HO
Ph
CO2Me
R1
O
H
O
N CO2MeR1
Ph
O
NR1
OLi
OMe
Ph
R2 X
O
NR1
CO2Me
R2
Ph
HN
HO
Ph
CO2MeR2
H2OAchiral aldehyde
LDA
N-benzylserine methyl ester
Seebach, D. et al. Helv. Chim. Acta. 1987, 70, 1194
21
First Total Synthesis
MeO2C
HNOH
Ph
OH
O
N
Ph
OTBS
O
N
PhCO2Me
OTBS
O
N
PhCHO
OTBS
1. TBSCl, Im,
2. TsOH, (CH2O)n,
LiBH4/THF MeOH
Swern
85%
92%
OH
NH
O
O
SNHAc
HO2C
Me
HOHO
22
First Total Synthesis
O
N
PhCHO
OTBS
O
O
O
N
Ph
OTBS
OO
MeHO
O
N
Ph
OTBS
OO
HOMe
2:1LDA, THF, -78 C 48%
30%
NH
O
O
SNHAc
HO2C
Me
HOHO
Pirrung-Heathcock anti-aldol gave poordiastereoselectivitey
23
Anti-Aldol Closed Transition States
O
O N OTBS
HO
O Li
H
ON O
OO
Li
O
OLi
O
N
Ph
OTBS
OH
OTBS
O
MeHO
O
N
Ph
OTBS
O
O
N
Ph
OTBS
OO
HOMe
+
24
First Total Synthesis
NO
O
Me
HOHO2C
NH
OMe
HO CO2H
SH
HSNHAc
O O
NO
O
Me
HOHO
NH
OMe
HO O
SNHAc
HO2C
HO
O
N
Ph
OTBS
OHO
Me
O1. H2/Pd-C2. 5% HF
1. Swern 2. NaClO2
78% 69%
BOPCl, Et3N
2. Pd(Ph3P)3 Et3N, HCO2H
1. H+95%
66%HO
HS
NH
O
O
SNHAc
HO2C
Me
HOHO
25
Drawbacks to Synthesis
Poor diastereoselectivity Needed to upscale to pursue biological studies
O
N
Ph
OTBS
OO
Me
HO
O
N
Ph
OTBS
OO
HO
Me
NH
OMe
HO O
SNHAc
HO2C
HO
H2N
HO
CO2Me
+2:1
26
Revised Aldol Reaction
O
NPh
CHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
N
Ph
OTBS
OO
MeHO
O
N
Ph
OTBS
HO OMeO
Me
O
N
Ph
OTBS
OO
HOMe
O
N
Ph
OTBS
HO OMeO
Me
2:1
LDA
9:1
ORIGINAL ROUTE
REVISED ROUTE
+
+
27
Revised Aldol Reaction
O
NPh
CHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
N
Ph
OTBS
OO
MeHO
O
N
Ph
OTBS
HO OMeO
Me
O
N
Ph
OTBS
OO
HOMe
O
N
Ph
OTBS
HO OMeO
Me
2:1
LDA
9:1
ORIGINAL ROUTE
REVISED ROUTE
+
+
Corey, E.J. et al. J. Am. Chem. Soc. 1998, 120, 2330
28
Magnesium Catalyzed Anti-Aldol
NO
O
Si MeMe
But
O
H
MgI
BOTTOM
TOP
Top face is favoured for attack of nucleophile
Bottom face is shielded by Benzyl and OTBS
NU
NU
29
Open Transition State Aldol
MgI
MgI
OO
N
OH
N
OH
N
OOTBS
CO2Me
Me
HON
OOTBS
CO2MeHO
Me
SYNCLINICAL ANTIPERIPLANAR
Anti-aldol Syn-aldol9:1
Me
HO
OMe
SiMe
MeMe
MeH
OMeOSi
Me MeMe
30
Improvements to Synthesis
Doubly diastereoselective aldol Synthesis of lactacystin in kilogram quantities Quantity allowed further biological investigation
NH
OMe
HO O
SNHAc
HO2C
HO
H2N
HO
CO2Me
O
N
Ph
OTBS
HO OMeO
Me
O
N
Ph
OTBS
HO OMeO
Me
+ 9:1
31
SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
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
NH
O
Me
O
SNHAc
HO2C
HO
NH
O
O
SNHAc
Me
HOHO
HO2C
Compound Proteasome Inhibition
Inactive
Inactive
NH
O
Me
O
SNHAc
HO2C
HO
ActiveHO
Lactacystin
OH cis to carbonylnecessary
33
Initial SAR Studies
NH
O
Me
O
OH
HO
NH
O
Me
Compound Proteasome Inhibition
Inactive
More Active
NH
O
Me
O
SNHAc
HO2C
HO
ActiveHO
HO
OO OH
Lactacystin
Electrophilic carbonylessential
34
Mechanistic Studies: In Vitro
Dick, L. et al. J. Biol. Chem. 1996, 271, 7273.
NH
O
HOHO
O
SNHAc
HO2C
Me pH 8, H2O NH
O
HOHO
O
OHMe
HSNHAc
CO2H+
35
Mechanistic Studies: In VitroA
238
(nm
)
Time (min)
NH
O
HOHO
O
SNHAc
HO2C
Me pH 8, H2O NH
O
HOHO
O
OHMe
HSNHAc
CO2H+
36
Mechanistic Studies: In Vitro
NH
O
HOHO
O
SNHAc
HO2C
Me pH 8, H2O NH
O
HOHO
O
OHMe
HSNHAc
CO2H+
A23
8 (n
m)
Time (min)
Not First order kinetics Suggests intermediate involved
37
Mechanistic Studies: Hypothesis
Is -Lactone an intermediate ? Increasing [NAC] will decrease rate of hydrolysis
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
OO
OH
NAC
HSNHAc
CO2H
NH
O
O
OH
HO
Me
HO+
38
Effects of [NAC] on Rate of Hydrolysis
2.0
1.5
1.0
0.5
0.00.1 1 10
k hyd
rol y
sis
(x 1
0-4
s- 1)
[NAC] (mM)
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
OO
OH
NAC
HSNHAc
CO2H
NH
O
O
OH
HO
Me
HO+
Addition of NAC impedesrate of hydrolysis
39
HPLC Detection of -Lactone
Ab
sorb
an
ce
0.00
0.05
0.10
1.01.21.4
5 10 15 20 25 30
Retention Time (min)
-Lactone
NH
O
Me
OO
OH
HSNHAc
CO2H
NH
O
O
OH
HO
Me
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
+
40
Mechanistic Studies
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
HO OHO
OH
NH
O
OH
Me
OO
INHIBITION
NO INHIBITION
INHIBITION
NO INHIBITION
pH 8
pH 6.3
NO -Lactone
pH 8 and pH 6.3
pH 8 and pH 6.3
41
Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HSNHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
42
Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HSNHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
43
Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HSNHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
+
Can Glutathione react with -Lactone to give a thioester adduct ?
Dick, L. et al. J. Biol. Chem. 1997, 272, 182.
44Retention Time (min)
Lactathione Formation In Vitro Confirmed
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
Glutathione Lactathione
+
Glutathione + -Lactone
Glutathione
-Lactone
45
In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
46
In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
47
In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
48
HPLC Analysis of Cell Extract
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin
-Lactone
Lactacystin
HPLC
HPLC
HPLCNH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
49
Fate of Lactacystin In Vivo : 2 Possibilities
CELLNH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin is impermeable to cell membrane
Lactacystin
50
Fate of Lactacystin In Vivo : 2 Possibilities
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
Conversion in cells occurs too rapidly for lactacytin to be detected
Lactacystin Lactathione
OR
CELLNH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin is impermeable to cell membrane
Lactacystin
51
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
52
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
53
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
54
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Results suggest that Lactacystin Is impermeable to cell membrane
Washed cells
Cell lysate
55
Mechanism of Action: Role of -Lactone
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
Lactacystin -Lactone
-Lactone is the active inhibitor Only -Lactone is permeable to cell membrane?
56
Hydrolysis of Lactacystin vs -Lactone
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
OHMe
HOHO
Lactacystin
Time (min)
-L
acto
ne]
57
-Lactone] Outside the Cell
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
OHMe
HOHO
- Lactone
Lactacystin
Hydrolysis of -Lactone is slower when starting with Lactacystin
Time (min)
-L
acto
ne]
58
Lactathione Accumulation in Cells
Lactathione accumulation is slower in Lactacystin treated cells
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
CELLS
NH
O
OH
Me
OO
NH
O
O
S
Me
HO
OCO2H
NH2
O NH
CO2H
HO
CELLS
Time (min)
[La
cta
thio
ne]
59
Mechanism of Action: Conclusions
Hydrolysis of -Lactone
- Lactone
Lactacystin
Time (min)
-L
acto
ne]
Lactathione Accumulation
- Lactone
Lactacystin
Time (min)
[La
cta
thio
ne]
Extracellular [-Lactone] Intracellular [Lactathione]
CONCLUSION
60
Mechanism of Action: Summary
NH
O
O
SNHAc
O2C
Me
HOHO
NH
O
OH
Me
OO
NH
O
O
OMe
HOHO
NH
O
OH
Me
OO
-GSH
+GSH
NH
O
O
S
Me
HO
OCO2
NH2
O NH
CO2H
HO
INSIDE CELL
OUTSIDE CELL
MEMBRANE
O
O
61
SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
OH and carbonyl are cis -Lactone formation is necessary for activity
62
SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
C9
C7
63
Synthesis: C9 Analogues
MeS CO2Me
Me CO2MeCO2HCO2Me
MeS
Me
N
OPMB
OHTBSO
CO2MeMe
MeSN
O
MePMB
TBSO
CO2MeCHO
N
O
O CO2MeH
PMB
Me
MeS
N
OPMB
HO
CO2MeOH
Me
MeS
PLE, PH 7
62%
1. (COCl)2 DMF2. PMB-NHCH2CO2Me, Et3N3. LDA,
92%
1. Formalin, DBU2. NaBH(OAc)3
86%
1. PivCl, Pyridine2. TBSOTf3. NaOMe,MeOH
77%
1. Raney Ni,2. Dess-Martin
78%
95%ee
99%ee
NH
O
Me
OO OH
Corey, E. J. et al. Angew. Chem. Int. Ed. 1998, 37, 1676.
64
SAR of C9 Analogues
N
OPMB
CHO
MeCO2Me
TBSO
N
OPMB
R
Me
TBSO
CO2Me
HO
NH
O
O O
R
OH
MeRMgBr,TMSCl
4 Steps
R
OH
OH
H
OH
OH
Entry Rel. kinhibition
1
2
3
4
5
1
0.006
0.003
Inactive
OH
0.095
Isopropyl group isalready optimum
65
SAR of C7 Analogues
O
N
PhCHOOTBS
R
OTMS
OMe O
N
Ph
OTBS
CO2Me
RHO
NH
O
R
O OOH
+
9 Steps MgI2
R
Me
H
Et
PHCH2
Entry Rel. kinhibition
1
2
3
4
5
1
0.15
2.18
2.33
0.73
nPr
Activity Increased with larger groups at C7
66
SAR Studies of Lactacystin
NH
O
O
SNHAc
HO2C
HOHO
Larger groups
EssentialElectrophillicCarbonyl
Essential
67
-Lactone : Important Feature for Activity
NH
O
OH
Me
OO
-Lactone
NH
O
OHOO
Salinosporamide A
Cl
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
H2N CO2Me
MeHO
MeOO
Cl
O
ClO N
Me
CO2Me
HO
OBn
PMB
MeOO
N CO2Me
Me
MeO
HN
HO
CO2Me
Me
OBn
57%
1.
2. pTsOH,
1. LDA, THF-HMPA ClCH2OBn2. NaCNBH3
62%1. TMSCl
2. , iPr2NEt,
3. H+
96%
NH
O
OHOO
Cl
Corey, E.J. et al. J. Am. Chem. Soc. 2004, 126, 6230
69
Synthesis of Salinosporamide A
O N
Me
CO2Me
HO
OBn
PMB
NOCO2Me
OBn
O
PMB
Me
NO
Me
CO2Me
OBn
OH
PMB
O N
Me
CO2Me
OBn
PMB
O
NO
OH
CO2Me
OBn
Me
PMB
Dess-Martin [O]
96%
Quinuclidine, DME90%
9:1
BrCH2Si(CH3)2ClEt3N, DMAP
95%
Si(Me)2CH2Br
NH
O
OHOO
Cl
70
Synthesis of Salinosporamide A
NOCO2Me
OBn
O
PMB
Me
Si(Me)2CH2Br
NO
PMB
OSi
MeMe
H Me
CO2Me
OH
H
ZnCl
NOCO2Me
OBn
PMB
OSi
MeMe
H Me
NO CHOCO2Me
PMB
OSi
MeMe
H Me
Bu3SnH, AIBN,
89%
1. H2/Pd-C2. Dess-Martin
88%
NH
O
OHOO
Cl
71
Synthesis of Salinosporamide A
NO
PMB
OSi
Me Me
H Me
CO2Me
OH
1. KF, KHCO3, H2O22. CAN
HNO
OHH Me
CO2Me
OH
HO
81%
1. LiOH2. BOPCl, Py3. Ph3PCl2, MeCN, Py
65%
NH
O
OHOO
Cl
Total synthesis of Salinosporamide A was achieved in 10%over 18 steps
72
Summary: Ubiquitin Proteasome Pathway
O
NH
Lys
O
NH
Lys
O
NH
Lys
Amino Acids Recycled Ubiquitin
UBIQUITIN
TARGET PROTEIN
73
Summary: Synthesis of Lactacystin
O
N
PhCHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
N
Ph
OTBS
OO
Me
HO
O
N
Ph
OTBS
HO OMeO
Me
LDA
ORIGINAL ROUTE
REVISED ROUTE
NH
O
O
SNHAc
HO2C
Me
HOHO
74
Mechanism of Action
NH
O
O
SNHAc
O2C
Me
HOHO
NH
O
OH
Me
OO
NH
O
O
OMe
HOHO
NH
O
OH
Me
OO
-GSH
+GSH
NH
O
O
S
Me
HO
OCO2
NH2
O NH
CO2H
HO
INSIDE CELL
OUTSIDE CELL
MEMBRANE
O
O
75
Summary: Synthesis of Salinosporamide A
H2N CO2Me
MeHO
NH
O
OHOO
Cl
Synthesized in 18 steps, 10% overall yield
76
Summary: SAR Studies and Analogs
NH
O
O
SNHAc
HO2C
Me
HOHO
Can be a larger group Essential to form -Lactone Initially optimized
NH
O
OH
Me
OO
NH
O
OHOO
- Lactone
Salinosporamide A
NH
O
OHOO
MLN-519Phase 1
Cl
Lactacystin
77
Acknowledgements
Dr. OgilvieLivia AumandAlison LemayMathieu LemayMatt ClayMy Family and FriendsAnd You!
