Bifunctional Molecular Probes for Activity-Based Visualization of Quinone-Dependent Amine Oxidases

Article abstract of DOI:10.1002/chem.201804247

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.

Full text of DOI:10.1002/chem.201804247

A Journal of
Accepted Article
Title: Bifunctional Molecular Probes for Activity-Based Visualization of
Quinone-Dependent Amine Oxidases
Authors: Ashley A Burke, Luke Barrows, Maria J Solares, Alexander D
Wall, and Charles Jakobsche
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201804247
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10.1002/chem.201804247
Chemistry - A European Journal
Title
Bifunctional Molecular Probes for Activity-Based Visualization of Quinone-Dependent
Amine Oxidases
Authors
Ashley A. Burke, Luke Barrows, Maria J. Solares, Alexander D. Wall, Charles. E. Jakobsche*
Contact
Clark University, Carlson School of Chemistry & Biochemistry, Worcester MA 01610,
email: cjakobsche@clarku.edu
Graphic Abstract
Table of Contents Text
The design, synthesis, and evaluation of molecular probes to visualize quinone-dependent
amine oxidase enzymes in an activity-dependent manner are described. These probes and
visualization strategy will enable activity-based protein-profiling experiments for this
family of amine oxidase enzymes.
Key Words
Arenes
Immunoassays
Inhibitors
Protein Modifications
Proteomics
Abstract
The design, synthesis, and evaluation of two bifunctional molecular probes that can
be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent
This article is protected by copyright. All rights reserved.

10.1002/chem.201804247
Chemistry - A European Journal
manner are described. These probes use alkylhydrazines to irreversibly bind the target
enzymes, which can then be visualized with either western blotting or in-gel fluorescence.
The results show that the western blotting readout, which utilizes commercially available
anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger
readout than the fluorescein-based fluorescence readout. This visualization strategy can be
used to measure the potency of enzyme inhibitors by selectively visualizing the active
enzyme that remains after treatment with an inhibitor. Looking forward, this probe
molecule and visualization strategy will enable activity-based protein-profiling
experiments, such as determining inhibitor selectivities within full proteome mixtures, for
this family of amine oxidase enzymes.
Main Body
An interesting subfamily of amine oxidase enzymes utilizes 1,2-benzoquinone
cofactors along with copper ions to enable their catalytic function. One member of this
subfamily that has received substantial attention as an exciting medical target is lysyl
oxidase (LOX).1,2,3,4,5 LOX's enzymatic activity has been correlated with the metastatic
potential of several types of cancer6,7,8,9,10 as well as with stiffening of connective tissue,11
myocardial fibrosis,11 chronic liver disease, 12 and hepatic fibrosis. 13 Other LOX-like
enzymes have also been identified as potential cancer targets.14,15 Like other amine
oxidases, this subfamily of quinone-dependent amine oxidases catalyze the oxidation of
amines into aldehydes. These enzymes can be irreversibly, yet non-selectively inhibited by
hydrazine-containing small molecules,16 with phenylhydrazine having been studied most
extensively.17 Other inhibitor structures such as β-aminopropionitrile have also been
studied.18,19 Structurally, the quinone cofactors contained within these enzymes, which are
formed from post-transnationally oxidized tyrosine residues, are often topoquinones (X =
OH in Figure 1 top left), but other variations such as lysyltyrosylquinones (X = a crosslinked
lysine sidechain) are also known.17 The work in this paper focuses on the lentil seedling
diamine oxidase enzyme (LSDAO), which is a typical representative of the topoquinone
subfamily, as well as the LOX enzyme, which utilizes the lysyltyrosylquinone cofactor.
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Chemistry - A European Journal
A
Molecular Probe Structure and Concept
Amine Oxidase
O
X
O
Readout
Domain
H
N
+
N
H
O
N
H
H2N
Tyr
O
3
3
1 (Readout = Fluorescein)
2 (Readout = 2,4-Dinitrophenyl)
LSDAO: X = OH
LOX: X = Lys–εNH
Amine Oxidase
X
OH
O
Readout
Domain
N
N
H
O
N
H
Tyr
N
3
B
Readout Domains
OH
O
O
NO2
Readout
Domain
=
or
N
H
N
H
O
N
H
OH
NO2
O
Visualize by
In-Gel Fluorescence
Visualize by
Western Blotting
Figure 1. Structures and overall concept for visualizing amine oxidase enzymes with
molecular probes 1 and 2.
Herein, we report the design and evaluation of molecular probes that can covalently
attach to quinone-dependent amine oxidases and enable their visualization. Molecular
probes 1 and 2 (Figure 1) each contain an alkylhydrazine group to bind the target enzymes.
Prior work, which includes X-ray crystallography20 and sequencing analysis17 of covalent
enzyme–inhibitor adducts, has shown that hydrazine-containing small molecules
covalently bind this family of enzymes by the general mechanism shown in Figure 1A. The
probes also contain flexible PEG-based linker regions and readout domains. We
hypothesized that probe 1, which contains a fluorescein readout domain, would enable
direct in-gel fluorescence visualization of this family of enzymes, and that probe 2, which
contains a dinitrophenyl readout domain, would enable antibody-based readouts such as
western blotting. While biotin could have been another option for a western blotting
readout domain, due to our previous success using the dinitrophenyl group as an antigen,21
we chose to continue exploring its utility for the current objectives.
The design of probe molecules 1 and 2 enables convergent approaches for their
syntheses via intermediate 7 (Scheme 1). The synthesis of key intermediate 7 begins with
an acid-promoted ring opening of ε-caprolactone (3), then uses a Mitsunobu-like coupling
with di-tert-butylazodicarboxylate (5) to directly install the doubly Boc-protected
hydrazine functionality,22 and finally reveals the carboxylic acid functionality via base-
promoted hydrolysis. Dinitrophenyl probe molecule 2 can be accessed by connecting acid 7
to amine 8 under standard peptide coupling conditions and then deprotecting the
hydrazine group with trifluoroacetic acid. The requisite amine 8 can be easily formed by
nucleophilic aromatic substitution of 1-chloro-2,4-dinitrobenzene (12) with diamine 13
under simple and mild conditions. Compared to similar biotin-containing small molecules,
the dinitrophenyl-containing molecules generally have reduced polarity and better
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10.1002/chem.201804247
Chemistry - A European Journal
solubility in organic solvents, which makes them somewhat more amenable to the process
of organic synthesis.
A
Synthesis of Probe Molecule 2
O
Boc
Boc
N
O
O
O
MeOH / H2SO4
74% y
5 (1 equiv)
LiOH
O
N
HO
OMe
OH
OMe
BocHN
BocHN
3
3
53% crude yield
46% y
(2 steps)
3
4
6
7
8 (1.5 equiv, crude)
EDC / HOBt
NO2
NO2
Boc
N
O
O
N
TFA
H
N
N
H
N
H
N
H
O
O
3
BocHN
TFA • H2N
H
3
3
3
87% y
NO2
76% y
NO2
9
2
B
Synthesis of Probe Molecule 1
OH
1. EDC / HOBt (12% y)
2. LiOH (quant)
Boc
O
O
O
O
O
H
N
N
+
N
H
N
H
O
OH
N
H
CF3
H2N
O
TFA • H2N
BocHN
O
O
3
3
3
3
3. NHS-Carboxy-
fluorescein (17% y, 2 steps)
4. TFA (74% y)
OH
7
10 (2 equiv)
1
Synthesis of Building Blocks
C
NH2
H2N
O
NO2
3
O
O
O
O2N
Cl
NO2
Br2
13
H
N
N
O
O
N
H
H2N
O
3
N
O
N
H
86% y
O
85% mass recovery
NO2
O
11
5
12
8
Scheme 1. Synthesis of probe molecules 1 and 2. See supporting information for detailed
procedures.
Fluorescein-containing probe molecule 1 can be synthesized by a similar strategy
(Scheme 1B). Acid 7 can be connected to amine 10, the trifluoroacetamide protecting group
can be hydrolyzed under basic conditions, the 5(6)carboxyfluorescein group can be
attached via its NHS-ester derivative, and the hydrazine group can be deprotected under
acidic conditions. Although the yield of this second sequence was low, sufficient material
was produced to enable its evaluation. Unfortunately, in contrast to dinitrophenyl probe
molecule 2, which is fully stable for use and long-term storage, fluorescein probe molecule
1 does undergo some autodecomposition. Preliminary analysis by LC/MS suggests that
over the course of days to months, depending on the storage conditions, the fluorescein
group may undergo redox chemistry23 with the hydrazine group to form a hydrazone,
which can then undergo various subsequent decomposition pathways such as hydrolysis or
elimination (Supplementary Figure S3 and accompanying description).
Initial experiments with probe molecules 1 and 2 show that both can be used to
visualize LSDAO, but that the dinitrophenyl probe and its western blotting system produce
stronger signals than the direct fluorescence readout from the fluorescein-containing
probe molecule. In a typical western blotting experiment, 50 ng of LSDAO are incubated
with 5–10 µM of dinitrophenyl probe molecule 2 for 10 minutes at 37 °C. These conditions
were selected based on our previous analysis of the kinetic of inhibition of this enzyme
with simple alkylhydrazine inhibitors.16 Lower concentrations of the probe such as 0.5 µM
can also be used, but the resulting signals are lower in intensity (Supplementary Figure S5).
The labeled protein is then run through an 8% SDS-PAGE gel, blotted onto a nitrocellulose
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10.1002/chem.201804247
Chemistry - A European Journal
membrane, treated with anti-nitrophenyl primary antibody followed by HRP-labeled
secondary antibody, and visualized by chemiluminescence with ECL substrate. 50 ng
quantities of the enzyme are readily visualized, and, as anticipated, negative control
experiments without enzyme or without the probe molecule produce no signals (Figure
2A). Lower quantities of LSDAO, as low as 1 ng, can be visualized, and the readout intensity
scales with concentration (Figure 2B). With fluorescein-containing probe molecule 1, on
the other hand, 135–270 ng of LSDAO can be visualized by in-gel fluorescence, but even
with these larger amounts of enzyme, the resulting signals are not as strong as those
obtained with the western blotting system (Figure 2C). The lower signal intensity,
however, does not seem to result from inefficient binding of probe molecule 1 to LSDAO
because this probe molecule is able to fully inhibit the catalytic activity of LSDAO, as
determined by a standard enzyme-activity assay (Supplementary Figure S4). These results,
combined with the partial instability of fluorescein probe 1 noted above, cause us to favor
the western blotting system with dinitrophenyl probe 2, which is what is used in the
subsequent experiments.
A
Western Blotting
C
In-Gel Fluorescence
no
enzyme
50
ng
no
probe
135
ng
135 270 270
ng
ng ng
B
Concentration-Dependent Visualization of LSDAO
50 ng 35 ng 20 ng 10 ng
5 ng
2 ng
1 ng
Figure 2. Visualization of LSDAO enzyme with probe molecules 2 and 1. A. 50 ng LSDAO, 9
µM dinitrophenyl probe 2, 30 seconds of imaging by chemiluminescence. A. 1–50 ng
LSDAO, 6 µM dinitrophenyl probe 2, 30 seconds of imaging by chemiluminescence. C. 135–
270 ng LSDAO, 180 µM fluorescein probe 1, 10 seconds of imaging by fluorescence.
To show that this system can selectively visualize enzymes that are catalytically
active, as opposed to inactive or inhibited, we analyzed the effect of pretreating enzyme
samples with inhibitors prior to visualization. Since irreversible binding of an inhibitor to
the enzyme's quinone cofactor would prevent the probe molecule from being able to
subsequently bind, we hypothesized that this blotting system would be able to indicate that
an enzyme sample had become inhibited by the disappearance of the readout signal.
Indeed, this disappearance of readout signal can be easily observed by pretreating LSDAO
with either of three known inhibitors: alkyhydrazine 14, acyl hydrazide 15, or
semicarbazide 16 (Chart 1), which we have previously studied as irreversible inhibitors of
quinone-dependent amine oxidase.16 A 4-minute pretreatment of the enzyme with 2, 30, or
800 µM of the alkyl inhibitor or 800 µM of either the acyl or semicarbazide inhibitors
completely blocks the readout signal; treatment with only 0.1 µM of the alkyl inhibitor or
30 µM of the acyl or semicarbazide inhibitors reduces, but does not fully block the signals;
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10.1002/chem.201804247
Chemistry - A European Journal
and treatment with only 2 µM of the acyl or semicarbazide inhibitors does not produce an
observable decrease (Figure 3A). An important control experiment, in which no decrease in
readout is observed when the enzyme is treated first with probe molecule 2 and then with
an inhibitor, shows that the probe molecule binds the enzyme irreversibly rather than
forming a reversible equilibrium (Supplementary Figure S6). An additional control
experiment shows that denaturing the target enzyme through heating also abolishes
labeling (Supplementary Figure S7).
Chart 1. Inhibitor Structures
O
O
NH2
NH2
NH2
N
H
N
H
N
H
N
H
heptylhydrazine 14
(alkyl)
hexyl hydrazide 15
(acyl)
butylsemicarbazide 16
(semi)
A
Activity-Based Visualization of LSDAO
none
0.1 µM
2 µM
30 µM 800 µM
Inhibitor
alkyl (14)
acyl (15)
semi (16)
n.d.
n.d.
B
Inhibitor Potency against LSDAO
Measured by a Traditional Enzyme-Activity Assay
100
alkyl (14)
50
acyl (15)
semi (16)
0
0.1
1
10
100
1000
Inhibitor Concentration (µM)
Figure 3. Comparison of activity-based Western blotting of LSDAO versus prior results
obtained with a traditional enzyme-inhibition assay. A. 50 ng LSDAO, 4-min incubation
with inhibitor at 37 °C, then 10-min incubation with 6 µM probe 2, visualize by Western
blotting. B. Previously reported data measuring the concentration-dependent inhibition of
LSDAO with a traditional assay (Reference 16). 4-min incubation with inhibitor at 37 °C
before analysis.
In these experiments, the extent to which the readout signal disappears correlates
with the extent to which the enzyme is inhibited by each inhibition condition. We have
previously used a traditional enzyme-activity assay24 to measure the inhibition of LSDAO
by these inhibitors (data reproduced in Figure 3B for ease of comparison),16 and we now
show the correlation of these data to the new results from the activity-based blotting assay.
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The concentrations of inhibitors that fully block the visualization signals are concentrations
that fully inhibit the enzymatic activity; the concentrations that produce negligible changes
to the visualization signals are concentrations that produce <25% inhibition of the enzyme;
and the concentrations that produce partial reduction of the visualization signals are
concentrations that produce 40–80% inhibition of the enzyme (Figure 3A versus 3B). Thus,
the blotting experiments are able to provide information about the potency of each
inhibitor, and the validity of these conclusions is supported by their consistency with data
from a traditional activity assay.
This visualization system can also be used to visualize LOX. Using dinitrophenyl
probe molecule 2, the western blotting procedure can clearly visualize 150 ng of LOX
(Figure 4A, left column). Compared to visualizing LSDAO, however, larger amounts of LOX
are needed, and the signals are not as intense. These differences may result from the fact
that LOX is an unstable enzyme and much of the isolated material is not catalytically
active—indeed, the in-house isolated material becomes completely inactive within a week
of storage—or from LOX's slower rate of enzymatic activity and lower affinity for
hydrazine-type inhibitors compared to LSDAO. To inhibit LOX, higher concentrations of
alkyl inhibitor 14 or acyl inhibitor 15 are needed; 13 µM concentrations of the inhibitors
only minimally reduce the output signal, but 330 µM concentrations show a more
substantial effect (Figure 4A). Also, it can be seen that acyl inhibitor 15 more significantly
reduces the output signal versus equivalent concentrations of alkyl inhibitor 14. As with
the LSDAO data, these results match well with prior enzyme-inhibition data that were
obtained with a traditional enzyme-activity assay (Figure 4B).16 These results show that
probe molecule 2 and this visualization system have the versatility to be able to report on
various quinone-dependent amine oxidase enzymes that encompass both the topoquinone-
type and lysyltyrosylquinone-type cofactors.
A
Activity-Based Visualization of LOX
none
13 µM 330 µM
Inhibitor
acyl (15)
alkyl (14)
B
Inhibitor Potency against LOX
Measured by a Traditional Enzyme-Activity Assay
100
acyl (15)
alkyl (14)
50
0
0.1
1
10
100
1000
Inhibitor Concentration (µM)
Figure 4. Comparison of activity-based Western blotting of LOX versus prior results
obtained with a traditional enzyme-inhibition assay. A. 150 ng LOX, 4-min incubation with
inhibitor at 37 °C, then 10-min incubation with 100 µM probe 2. B. Previously reported
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10.1002/chem.201804247
Chemistry - A European Journal
data measuring the concentration-dependent inhibition of LOX with a traditional assay
(Reference 16). 4-min incubation with inhibitor at 37 °C. before analysis.
The results reported herein expand the field of activity-based protein profiling
(ABPP), which is a growing branch of chemical biology. ABPP experiments typically aim to
use molecular probes to enable the study of a family of proteins, to selectively detect
proteins that are functionally active, and to use comparative analyses to study how the
levels of these functionally-active proteins vary between different samples or conditions.25
Pioneering work in this field by the Cravatt group focused on using fluorophosphonate-
containing bifunctional molecular probes to visualize the family of serine protease
enzymes.26,27 These researchers and others have continued to expand the field, for example
by developing molecular probes to report on various other families of enzymes,28,29, 30, 31 by
using probes to identify proteins whose active forms are upregulated in diseases states
such as in metastatic versus benign cancers,32,33 by using these probes to visualize how
inhibitors affect whole families of enzymes within full proteome mixtures in order to
inform inhibitor discovery,34,35 and by combining with photoaffinity strategies to identify
protein–protein interactions.36 Similar molecular probes and strategies have also been
used to study families of post-transitionally modified non-enzymatic proteins. The family of
carbonylated proteins, for example, can be tagged by arylhydrazine probe molecules.37,38
Dinitrophenyl probe molecule 2 and the visualization strategies that we report herein will
enable a variety of ABPP-type experiments to be performed on the family of quinone-
dependent amine oxidases.
Acknowledgements
This work was enabled by funding from Clark University, the James and Ada
Bickman Fund (MJS), The Gustaf H. Carlson Fund (AAB), The Sherman Fairchild Scholars
Program (LB), and undergraduate fellowships from PCI Synthesis Inc. (LB, MJS, AAB). We
are grateful to Prof. Fred Greenaway for his assistance with enzyme purifications and
assays as well as for helpful discussions.
Conflict of Interests
The authors declare no conflicts of interest.
Supporting Information
1
Synthetic procedures, compound characterization, copies of H NMR, 13C NMR and
LCMS spectra, detailed procedures for assays.
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