High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
Timothy S. Schaefer
David H. Stewart
Robert M. Umek
and Jacob N. Wohlstadter
®
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High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
1
Abstract
The latent transcription factor Stat3 (signal transduction and activator of transcription) has been implicated in contributing to
neoplasia because it is aberrantly activated in numerous tumors and tumor-derived cells and is widely regarded as a key
target for therapeutic intervention. Upon phosphorylation on a single tyrosine residue (Y705), the Stat3 protein forms dimers
via mutual SH2 domain/pY705 interactions, a requisite step for nuclear translocation, DNA binding and transcriptional
activation. We have developed an assay that mimics the interaction between the Stat3 SH2 domain and pY705 using
immobilized protein decorated with a phosphopeptide encompassing this tyrosine residue and recombinant Stat3 protein. The
assay is robust (signal to background values >8) and reliable (Z'=0.6) and binding constants on the order of 157nM have
been obtained. It is being employed to identify small molecules that interfere with Stat3 dimerization which may hold promise
as cancer therapeutic agents.
®
A division of Meso Scale Diagnostics, LLC.
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
2
MSD MULTI-ARRAY™ and MULTI-SPOT ® Plates
Instrument Features
• Highly sensitive imaging detection systems
• Single and multiplex plate formats
• SECTOR 6000 instrument designed for high-throughput screening (HTS)
• Rapid read times
• SECTOR 6000 or SECTOR PR instruments ideal for assay development
• Electrochemiluminescent (ECL) technology
SECTOR PR
SECTOR Imager 6000
TM
TM
Plate Features
• Disposable Plates
• Carbon Electrodes with high binding capacity
• Suitable electrochemistry for ECL
• A variety of surface treatments, array preparations and coatings are available
®
A division of Meso Scale Diagnostics, LLC.
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
3
Electrochemiluminescence (ECL)
Measured signal is light
LIGHT
Luminescence
Emitting Light
*Ru(bpy)3
2+
-H+
TPA .
Chemi-
Chemical Energy
Ru(bpy)32+
Ru(bpy)33+
TPA .+
TPA
Electroe-
e-
Electrode
Electrochemically Initiated
Ruthenium (II) tris-bipyridine-(4-methylsulfone) NHS ester (MSD SULFO-TAG™ label)
S O 3Na
• Selective
S O 3Na
• Convenient chemistry
• Robust, stable
• Few interferences
N
N
S O 3Na
N
O
O
R u 2+
N
O
O
N
N
N
S O 3Na
®
A division of Meso Scale Diagnostics, LLC.
• Size, MW:
~1200 daltons
• Stability:
Years
• Solubility:
Aqueous, DMSO
• Functionality:
Hydrophilic
• Specificity:
High
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
4
Stat3 Proteins
DNA-B
SH2
Y
S
• Src homology 2 (SH-2) containing transcription factor.
• Activated by phosphorylation on a single tyrosine (Y).
• Form dimers via SH-2/phosphotyrosine interaction and rapidly translocate to the nucleus.
• Transcriptional activity can be augmented by serine phosphorylation (S).
• Constitutively activated Stat3 in tumors increases the proliferative capacity of cells (increased c-myc message and
protein) and decreases apoptosis (increased expression of the anti-apoptotic gene bcl-XL).
Mechanisms of Stat3 Activation
Cytokine
Receptor
K
P
P
P
P
SH2
P
P
P
P
SH2
P
P
P
P
P
JAK
K
P
Lig
LIG
Receptor
Tyrosine
Kinase
P
SH2
5
SH2
P
®
A division of Meso Scale Diagnostics, LLC.
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
6
Depiction of the Stat3-SH2/phosphotyrosine Binding Assay
A
Stat3
SH2
P
P P
P
P P
P
BSA
Carbon Electrode
P
B
pY705 peptide
Y705 peptide
STAT3-sTAG
BSA-peptide conjugate
Schematic representation of Stat3 SH2/domain interaction assay. A. Shows the binding of dimer-incompetent recombinant Stat3
to immobilized BSA that has been decorated with a phosphopeptide encompassing tyrosine 705 of Stat3. B. Shows the
representative binding of sTAG-labeled Stat3 to the immobilized BSA peptide conjugates (Left, pY705; Right, Y705).
7
Binding of Stat3 to Stat3-Derived Phosphopeptide
40000
BSA-coupled phosphopeptides were deposited onto the
surface of a 384-well MULTI-ARRAY plate. MSD SULFO-TAGlabeled recombinant, dimer-incompetent Stat3 protein was
then added to the wells at the concentrations indicated.
The plate was incubated at room temperature for 4 h,
washed and MSD Read Buffer added. It was then imaged
on the SECTOR Imager 6000 reader.
S-B
30000
20000
KD 157nM
10000
0
0
250
500
750
1000
MSD SULFO-TAG-Stat3 (nM)
®
A division of Meso Scale Diagnostics, LLC.
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
8
Assay Stability in Read Buffer
8
pY705/Y705 Binding
7
6
5
4
3
2
1
0
0
10
20
30
40
60
Time following
Addition of MSD
Read Buffer (min)
MSD-SULFO-TAG-labeled recombinant Stat3 protein (150nM) was added to the well of a 384-well MULTI-ARRAY plate in 1x
Binding buffer. The plate was then incubated at room temperature for 4h. MSD Read Buffer added and the plates were imaged
on the SECTOR Imager 6000 reader at the times indicated above. The ratio of Stat3 binding to the phospho- and nonphospho-peptide are indicated.
9
DMSO Compatibility
pY705/Y705 Binding
10
8
6
4
2
0
0
1
2
3
4
5
6
8
DMSO Concentration (%)
MSD-SULFO-TAG-labeled recombinant Stat3 protein (150nM) was added to the well of a 384-well MULTI-ARRAY plate in 1x
Binding buffer containing DMSO at the concentrations indicated. The plate was incubated at room temperature for 4h, MSD Read
Buffer added and imaged on the SECTOR Imager 6000 reader. The ratio of Stat3 binding to the phospho- and non-phosphopeptide are indicated.
®
A division of Meso Scale Diagnostics, LLC.
High Throughput Assay to Screen for Inhibitors
of the Proto-Oncogene Stat3
10
HTS Capabilities of Stat3/Phosphopeptide Assay
30000
Signal
25000
20000
15000
10000
5000
0
200
400
600
800
1000
1200
1400
Homogeneous Assay Workflow
• MULTI-ARRAY plates were printed with BSA-coupled phosphopeptides (or control peptide)
• Incubate with MSD SULFO-TAG-labeled recombinant, dimer-incompetent Stat3 and incubated
at room temperature for 4 hr (Inhibitors could be added at this step)
• The plates were then imaged on the SECTOR Imager 6000 reader
Gray squares indicate binding to the non-phosphorylated Stat3 peptide; Red Squares indicate binding to pY705.
Signal/background values of 8 and a Z' score of 0.6 indicate that the Stat3/Phosphpeptide assay is amenable for use in high
throughput screening efforts.
11
Conclusions
Stat3 is constitutively activated in numerous types of cancers making it an important target for therapeutic intervention.
We describe an assay that assesses the interaction between the Stat3 SH2 domain and tyrosine 705 that mimics Stat3
dimerization, the key point in activation of the molecule.
The assay is robust, DMSO-tolerant, stable in Read Buffer making it amenable to high throughput screening.
It is being employed to screen for small molecules that interfere with the formation of Stat3 dimers.
These lead compounds could be used to block the function of Stat3 in tumors in which it is aberrantly activated.
®
A division of Meso Scale Diagnostics, LLC.