A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Marta Corcoran,
Robert M. Umek, Sapna Dharkar,
James L. Wilbur, George B. Sigal,
Eli N. Glezer, Hans A. Biebuyck
and Jacob N. Wohlstadter
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Abstract
We have developed a novel assay for protease activity using a new assay
platform developed by Meso Scale DiscoveryTM (MSDTM). This platform combines
array technologies and electrochemiluminescence detection to achieve ultra-fast,
highly sensitive assays. This poster presents an assay for matrix metalloproteinases
(MMPs) activity. Oligopeptides containing the cleavage sequence for a subset of
MMPs and bearing an electrochemiluminescent complex were immobilized on
carbon electrodes integrated into microtiter plates. Subsequent challenge of the
immobilized oligopeptides with the catalytic domain of MMP3 revealed catalytic
rate constants (Kcat/Km) in agreement with published values. The use of the
MMP3-specific inhibitor, NNGH, confirmed the specificity of cleavage. The assay is
robust using as little as 1 picomole of substrate, minimizing the cost of
substrate. Both the immobilized substrate and the quenched reaction are stable
over many hours facilitating easy adaptation to automation. This novel platform
will facilitate high-throughput discovery of protease-specific inhibitors.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Multi-Array Technology
TM
Unified technology platform with instruments, plates and reagents
for drug discovery.
Combines the power of microarrays with the sensitivity of
electrochemiluminescence.
96-, 384- and 1536 microplate formats.
Multi-Spot plates with high density arrays for multiplexing.
TM
Sector HTS Instrument: High resolution imaging detection and
robotic integration for HTS and large-scale proteomics.
TM
Sector HTS
Sector PR Instrument: Medium throughput benchtop reader for
assay development, cellular and molecular biology, research in
therapeutic areas, secondary screening, QC. Assays developed on
Sector PR port to Sector HTS.
TM
Sector PR
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Schematic of the Protease Assay
Protease
A
A
A
A
carbon electrode
A
A
carbon electrode
An oligopeptide, modified with biotin at one end and an electrochemiluminescence label (a derivative of Ruthenium (II)
tris-bipyridine, ) at the other, is immobilized to an avidin(A)-coated electrode. Cleavage of the peptide by a sequence
specific endopeptidase results in a decrease in the amount of label proximal to the surface and thus a decrease in
signal. The active, catalytic domain of MMP3 protease was purchased from CalBiochem.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Biotin-Dependent Immobilization & Enzyme-Specific Cleavage
of an Oligopeptide
A
102
Peptide
Alone
120
R2=0.99
102
102
Biotin+Peptide
102
10
2
0.1
1
MMP3 +phen
MMP8 +phen
100
Percent Substrate
Remaining (%)
Signal
102
Differential Cleavage of
Immobilized Peptide by
Distinct MMPs
B
Oligopeptide Immobilization on
Avidin-Coated Electrodes
10
100
80
MMP2 +phen
MMP8 kcat/km=875
60
MMP2 kcat/km=12210
40
MMP3 kcat/km=20560
20
0
0
25000
Peptide (nM)
50000
75000
TIME (sec)
A) A control experiment showed that immobilization of the peptide was biospecific (i.e. mediated by avidin/biotin
interactions). Dual-labeled oligopeptide was incubated above avidin-coated carbon electrodes at varying concentrations in
the absence (blue) or presence (green) of a vast molar excess of free biotin. Signal output increases linearly over the
concentration range studied and correlates with quantitative immobilization of the substrate.
B) One picomole of dual-labeled oligopeptide was immobilized on avidin-coated carbon electrodes and challenged with
5nM of each enzyme in the presence or absence of 10nM 1,10-phenanthroline. The reactions were conducted in MES
buffer (50mM MES pH6.0, 10mM CaCl2, 0.05% Brij35,1uM ZnSO4, 150mM NaCl).
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
The MMP3 Specific Inhibitor NNGH Exhibits Characteristic
Properties in the Assay
S1' Pocket
A
C
MMP3 Specific Inhibition by NNGH
Leu164
N
OGlu202
1.5×1006
H
O
Ala165
O
OMe
O
O
S N
H N
H O
His201
Pro210
(Hydrophobic)
B
S2' Site
O
Zn2+
His211
His205
S1' Site
Phe210
(Hydrophobic)
IC50 of NNGH vs MMP3
100
80
no enzyme
10nM MMP3
5nM MMP3
10nM MMP2
5.0×1005
0.0×10-00
1
10
100
1000
10000
60
% inhibition
SIGNAL
1.0×1006
40
Soluble=19nM
Immobilized=20nM
20
0
100000
NNGH (nM)
10
-1
10
0
10
1
10
2
10
3
10
4
NNGH (nM)
A) Immobilized, dual-labeled oligopeptide was challenged with MMP3 or MMP2 in the presence of varying concentrations of
the known, MMP3 specific inhibitor (c) N Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH). The signals
observed in the absence of inhibitor are plotted on the ordinate. Note that MMP3 is inhibited at higher concentrations,
while MMP2 is not.
B) The percent inhibition observed over a range of NNGH concentrations was examined for MMP3 cleavage of the duallabeled peptide either free in solution or immobilized to avidin-coated carbon electrodes. The IC50 values are in agreement.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Enzyme Kinetics are Unaltered by Substrate Immobilization
A
B
Soluble Peptide
100
Immobilized Peptide
100
1hr - 37 C
20hr - 37oC
1hr - 25oC
20hr - 25oC
80
60
40
1hr - 37oC
20hr - 37oC
1hr - 25oC
20hr - 25oC
80
% Substrate Remaining
% Substrate Remaining
o
60
40
20
20
0
0
0.1
1
10
100
0.1
Enzyme (nM)
1
10
100
Enzyme (nM)
The dual-labeled oligopeptide was incubated with active MMP3 enzyme either in solution (A) or after being immobilized
through binding to an avidin-coated carbon electrode followed by incubation with the enzyme (B). In A, intact peptides
and cleaved fragments were captured to the electrode after the cleavage reaction. The percentage of substrate remaining
is plotted as a function of enzyme concentration at 25oC and 37oC after 1hr or 20hrs. For both the soluble and
immobilized substrates, the rate of substrate consumption is linear over the range of enzyme concentrations studied.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Short Term Stability of Immobilized Peptide and the Terminated Reaction
Cleavage of Stored
Oligopeptide
A
40
Fresh
Wet
Dry
Stability of the Terminated
Reaction
100
Remaining
Percent of signal
30
Remaining
Percent Substrate
B
20
10
No enzyme
80
60
40
20
10nM Enzyme
0
0
4.0
25.0
0.1
1
10
100
37.0
Hours after addition of Assay Buffer
Temperature (oC)
A) Dual-labeled oligopeptide was immobilized on avidin-coated carbon electrodes and stored 24 hrs., wet or dry, at
various temperatures. Subsequently, the immobilized peptide on the stored and, for comparison, freshly prepared plates
was challenged with 20nm MMP3 for 2 hrs. at 25oC.
B) The stability of the terminated reaction was determined through quantification of the signal remaining over a 24 hr
period after the reaction was stopped.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for Sequence-Specific
Cleavage of an Immobilized Oligopeptide
Conclusion
A protease assay has been developed on a novel platform that utilizes
carbon electrodes arrayed in disposable microtiter plates.
Synthetic, dual-labeled oligopeptides, containing biotin and a ruthenium
metal chelate serve as substrate.
Immobilization of the peptide via the biotin to avidin-coated electrodes
does not alter enzyme kinetics.
As little as 1pMol of substrate and 5nM enzyme are sufficient.
The stabilities of the immobilized substrate and the terminated
reaction are compatible with high throughput screens for protease-specific
inhibitors.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM