Download Chemical methods for Dissecting Protease Function

Small Molecule Probes for in vivo Imaging
of Protease Function
Overview
O
HN
O
N
H
• Why Activity is Important
Biotin
H
N
O
O
O
H
N
N
H
O
OH
O
X
O
X= Arg, Leu, Asp, Asn, Gly
• Activity Based Probes (ABPs)
– What are they?
– How do they work?
• Applications of ABPs
– Biochemical profiling
• Caspases - Alicia Berger - Mol. Cell. 2006,
23:509-521
– Imaging
• Cathepsins - Galia Blum
– Chemical Genetics
• Protease function in human parasites Carolyn Phillips, Shirin Kapur
– Toxoplasma gondii
– Plasmodium falciparum
Activity Based Probes for Proteases
O
R
O
H
N
H
N
N
H
O
R
N
H
R
O
R
O
Peptide Substrate
O
Tag
R
O
H
N
RFG
N
H
O
R
Activity Based Probe
Activity Based Probes for Proteases
O
Tag
R
O
O
H
N
O
N
H
O
R
-S
R
Tag
O
R
O
H
N
N
H
O
Enz
R
S
Enz
AOMK
Cat K
Cat F
Cat C
Cruzain
Cat H
Cat S
Cat L
Papain
Cat V
Cat B
The qABP
GB11
GB111
GB17
GB117
Quenched Probe Strategy
Imaging in Live Cell Monolayers
GB111
GB111 + JPM
LysoTracker
LysoTracker + JPM LysoTracker
LysoTracker + JPM
Merge
Merge
Merge
GB117
Merge
GB117 + JPM
Blum et al. Nature Chemical Biology, 2005, 1, 203-209
Stability of Probes
GB111
Serum (Hrs):
Inhibitor:
– –
– +
1 4
– –
GB117
– –
– +
1 4
– –
GB136
– –
– +
1 4
– –
97
66
42
36
Cat B
Cat L
Cat L
28
20
Non quenched
Quenched
Quenched
Cathepsins and Cancer
•
Cathepsins have been linked to
cancer progression
– Cathepsin B - mainly in cell
culture models
•
Recently more complex mouse models have been tested
– RIP-Tag - Joyce et al. (2004) Cancer Cell, 5, 443-453
– PyMT - study with cat B -/- mice - Vasiljeva et al. (2006) Cancer
Research, 66, 5242
– Angiogenesis model - Cathepsin L - Urbich et al. (2005) Nature
Medicine, 22, 206-213.
– RIP-Tag/ Cat K/O - Joyce et al. (2006) Genes and Development,
20, 543
•
All support a role for cathepsins in tumor growth and angiogenesis
– Protease activity provided by tumor microenvironment not just
tumor cells
Multistage tumorigenesis in RIP-Tag
transgenic mice
Normal stage
(onc+)
Hyperplastic/
Dysplastic
stage
Angiogenic
stage
Tumor
stage
<4 wks
4-7 wks
5-12 wks
10-14 wks
100%
~50%
~10%
2-4%
Cathepsin activity profiling in vivo
Exocrine
pancreas
Normal
islet
Normal
islet
x200
Fluorescent cathepsin probe
Fluor cathepsin probe/DAPI
Exocrine
pancreas
Tumor
x100
Joyce et al. Cancer Cell (2004) 5, 443-53.
Tumor
Need for NIRF probes
NIRF Probe and Control
N
N
N
HN
N
O
HN
H
N
O
O
O
O
O
N
H
O
O
H
N
O
O
GB123
O
N
H
NH2
O
GB125
Georges von Degenfeld
Galia Blum
Cathepsin Activity in Tumor Cell Lines
C2C12 par C2C12 ras U87MG
‫ذ‬
+
‫ذ‬
+
‫ذ‬
+
MCF-7 MDA MB-435 MDA MB-231
‫ذ‬
+
‫ذ‬
+
‫ذ‬
+
98
*
66
42
36
28
20
14
Ex/Em 633/670
In vivo Imaging of Cathpsins
Baseline
2 hr
4 hr
8 hr
10 hr
12 hr
24 hr
500
450
MDA-MB# 435 231
X106
400
350
p/sec/cm 2
Baseline
MDA-MB# 231 435
2 hr
8 hr
12 hr
24 hr
In vivo Imaging of Cathpsins
GB123
Baseline
4.0
0.5 hr
5 hr
Signal to Noise
23 hr
4
3.6
3
2
3.2
GB123
1
0
0
MDA MB231 MFP
3.0
GB138
25
GB138
0
5
10
15
20
Hours after injection
1.0
GB123 GB125
20
2
1
0
1.5
1 min
15
3
2.0
MDA-MB-231
Before injection
10
7
6
5
4
2.5
MDA MB231 MFP
5
5h
22 h
25
Need for NIRF probes
Probe:
123 125
123 123
- +
acat B a cat L
Tumor Origin: MDA-MB 23 C2C12/ras
Local DMSO:
97
97

66
42
36
36
28
cat B
28
cat L
20
20
Ex/Em 633/670
In Vivo Images
66
42
Ex/Em 633/670
Imaging Drug Efficacy
Ki777
AMS28
Vehicle
Ki777
AMS28
Ki777
Vehicle
AMS28
1.0
0.9
0.7
x109
0.8
0.6
0.5
Base line
30 minutes
8 hours
Tumor Fluorescence
Ki777
Vehicle
9.0
4.0
8.0
5.0
4 hours
Ki777
12
3.5
3.0
AMS28
8.0
2.5
2.0
1.5
4.0
Vehicle
0.0
Ki777
Vehicle
1.0
x109
6.0
x109
7.0
p/sec/cm2/srX109
16
Quantification of Drug Efficacy
Tumor
Kidney
Liver
Conclusions
• Activity based probes can be used to monitor protease
ACTIVITY in complex proteomes
• Quenched activity based probes allow real time imaging of
protease activation
• Near Infrared fluorescent probes can be used to monitor
protease activity in live animals
– Potential applications
• Diagnostic imaging - fluorescent, PET
• Pharmacodynamic studies
• Drug efficacy studies
Acknowledgements
The Bogyo Lab
Galia Blum
Shirin
Arastu-Kapur
Alicia Berger
Carolyn
Phillips
Beth Ponder
Galia Blum
Daisuke Kato
Steven Verhelst
Kelly Sexton
Marko Fonovic
Alicia Berger
Carolyn Phillips
Shirin Arastu-Kapur
Masoud Sagadhiani
Fang Yuan
Beth Ponder
Mark Rice
NIMR Mill Hill
Mike Blackman
Sharon Yeoh
Stanford
Georges von Degenfeld
Helen Blau
The Burnham
Guy Salvesen
Jean Bernard Denalt
Sloane Kettering
Johanna Joyce
UCSF
Doug Hanahan
Jessica Pahler
Compound Libraries
James Powers
Bill Rousch
UVM
Gary Ward
Jeralyn Haraldsen
Funding
http://bogyolab.stanford.edu
Department of Defense - BCCOE, Sandler Family, Searle Scholars Fellowship, NIH Roadmaps NTCNP Grant,
Burroughs Wellcome Fund, NIH PPG