Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors

Article abstract of DOI:10.1002/anie.201201955

High profile: New activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action. Copyright

Full text of DOI:10.1002/anie.201201955

Angewandte
Chemie
DOI: 10.1002/anie.201201955
Proteomics
Activity-Based Probes for Studying the Activity of Flavin-Dependent
Oxidases and for the Protein Target Profiling of Monoamine Oxidase
Inhibitors**
Joanna M. Krysiak, Johannes Kreuzer, Peter Macheroux, Albin Hermetter, Stephan A. Sieber,*
and Rolf Breinbauer*
Dedicated to Professor Herbert Waldmann on the occasion of his 55th birthday
Activity-based protein profiling (ABPP) has become a power-
ful chemical proteomic technology allowing the dissection of
complex ligand–protein interactions in their native cellular
environment.[1] The application of small-molecule activity-
based probes to interrogate enzyme activity on the cell level
has led to the identification and functional characterization of
proteins involved in cancer,[2] signaling pathways,[3] microbial
pathogenesis and virulence,[4] host–virus interactions,[5] and
other biological processes. However, up to now most ABPP
studies have aimed at enzyme classes with well-established
catalytic mechanisms and nucleophilic active-site residues
participating in the formation of a covalent bond to activity-
based probes (e.g. serine hydrolases,[6] cysteine[7] and threo-
nine proteases[8]). Thus, one important challenge in ABPP is
expanding the pool of probe molecules to enzyme classes with
more complex catalytic activities such as kinases,[3,9] trans-
ferases,[10] and oxidoreductases[11] to extend the proteome
coverage. Here, we introduce unprecedented activity-based
probes for an important group of oxidoreductases, namely
flavin-dependent oxidases.
Flavin-dependent enzymes catalyze a diverse set of
reactions encompassing oxidations, monooxygenations, dehy-
drogenations, reductions, and halogenations, making them
indispensable for many cellular processes.[12] Among them,
flavin-dependent oxidases represent a complex subgroup that
oxidize a broad spectrum of molecules by the employment of
molecular oxygen as an electron acceptor.[13] Their intrinsic
structural diversity, multiplicity of accepted substrates, and
lack of conserved residues in the active site make them elusive
to functional annotation by established genomic, structural,
and proteomic analyses.[14] In contrast, ABPP could serve as
a powerful and simple alternative for global profiling of these
enzymes. We envisioned that selective activity-based probes
could be built on the simple principle of the binding affinity of
the oxidatively activated probes towards the flavin cofactor,
the only common and intrinsic feature of flavin-dependent
oxidases.[15]
We present here the development and biological evalua-
tion of a novel chemoproteomic strategy dedicated to flavin-
dependent oxidases which involves a “tag-free” approach for
in situ enzyme labeling within intact cells. Subsequent cell
lysis followed by click chemistry[16] results in the attachment
of the fluorescent tag which serves in the visualization of
enzyme activities by gel electrophoresis and fluorescence
scanning. A LC–MS-based platform finally reveals the
identity of labeled enzymes (Figure 1A).
[*] J. M. Krysiak, Prof. Dr. R. Breinbauer
Institute of Organic Chemistry, Graz University of Technology
Stremayrgasse 9, 8010 Graz (Austria)
E-mail: breinbauer@tugraz.at
J. Kreuzer, Prof. Dr. S. A. Sieber
Center for Integrated Protein Science CIPSM
Department of Chemistry, Institute of Advanced Studies IAS
Technische Universitꢀt Mꢁnchen
Lichtenbergstraße 4, 85747 Garching (Germany)
E-mail: stephan.sieber@tum.de
The designed ABPP methodology was examined using
a monoamine oxidase enzyme, a representative example of
flavin-dependent oxidases, to validate the new labeling
mechanism on a well-known target. Monoamine oxidases[17]
(MAO, EC 1.4.3.4) are flavin adenine dinucleotide (FAD)-
containing enzymes, localized in the mitochondrial outer
membrane, which catalyze the oxidative deamination of
several important neurotransmitters in the central nervous
system (CNS), including serotonin, norepinephrine, and
dopamine as well as xenobiotic amines. In humans, mono-
amine oxidases exist in two isoforms designated MAO A and
MAO B,[18] which are encoded by two distinct genes[19] on the
X chromosome and display unique substrate selectivities and
inhibitor sensitivities[18] although they share a high level of
sequence identity (70%).[19] The elucidation of the crystal
structures of isozymes MAO A[20] and MAO B[21] provided
detailed insight into structural differences in their active sites
Prof. Dr. P. Macheroux, Prof. Dr. A. Hermetter
Institute of Biochemistry, Graz University of Technology
Petersgasse 12, 8010 Graz (Austria)
[**] We thank Prof. D. E. Edmondson (Emory University, Atlanta (USA))
for the donation of monoamine oxidases, Prof. Dr. W. Berger
(Medical University of Vienna, Vienna, Austria) for the donation of
human cell lines, and Prof. Dr. R. Zimmermann (Graz University,
Graz, Austria) for use of his cell culture facility. We thank Mona
Wolff for excellent scientific support. J.M.K. was supported by the
Austrian Science Foundation (FWF) grant W901-B05 DK Molecular
Enzymology. S.A.S. thanks the DFG (SFB 749, FOR 1406) and ERC
(Starting Grant) for financial support. J.M.K. thanks Milagros
Aldeco, Dr. Alexandra Binter, Dr. Ute Stemmer, and Venugopal
Gudipati for helpful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201955.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://angewandte.org/open.
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argylamine group. Mitsunobu reaction
with 4-pentyn-1-ol allowed the attach-
ment of an alkyl linker carrying an
alkyne group under very mild conditions
to furnish the appropriate probes (Fig-
ure 2C). Structurally diverse products
were prepared through the use of differ-
ent substitution patterns at the amino
group and by the various lengths and
structures of the carbon skeleton between
the reactive group and arene ring.
To test whether the incorporation of
an alkyne handle into the pargyline and
deprenyl changes their MAO inhibition
potency, we determined IC50 values of the
synthesized probes in
a
continuous
Amplex Red/peroxidase-coupled assay
and compared them to the parent inhib-
itors (Figure S1 in the Supporting Infor-
mation). Fortunately, the structural modi-
fications introduced to the probes did not
Figure 1. A) Identification of a target protein by the use of ABPP. B) Labeling of activity-based
probes dedicated to monoamine oxidases (FAD-dependent oxidases).
accounting for distinct substrate and inhibitor specifici-
ties.[20,22] Moreover, it confirmed the covalent attachment[23]
of the isoalloxazine ring of the FAD cofactor through an 8a-
(S-cysteinyl) linkage to a cysteine residue.[24] On account of
their involvement in catabolism and the regulation of neuro-
transmitters, monoamine oxidases play an essential role in
normal brain development and function. It was found that
mutations in the MAO A gene are implicated in manifesta-
tions of aggressive behavior[25] while age-related increases of
expression levels of MAO A in the heart[26] and MAO B in
neuronal tissue[27] have been associated with the development
of cardiovascular[28] and neurodegenerative disorders,[29]
respectively. Hence, MAO inhibitors are clinically used for
the treatment of depression, Parkinsonꢀs disease, anxiety
disorders, and other mental diseases.[30]
We designed and synthesized a small set of novel activity
probes (Figure 2A) based on the structure of the known
irreversible MAO inhibitors pargyline and deprenyl (Fig-
ure 2B). These inhibitors have been used in the functional
and biochemical characterization of monoamine oxidases and
are useful drugs in clinical applications.[31,32] Both inhibitors
feature an N-propargylamine group which is essentially
involved in irreversible enzyme inhibition and the formation
of a stable covalent adduct, whose existence has been
unambiguously proven by crystal structures of MAO with
acetylenic inhibitors such as deprenyl, clorgyline, and rasagi-
line.[20,33] In the initial step of this inhibition mechanism, the
FAD cofactor catalyzes the oxidation of the amine group to
an iminium cation producing a reactive Michael acceptor,
which can be nucleophilically attacked by the N(5) atom of
the isoalloxazine ring leading to the formation of the covalent
adduct. We anticipated that activity-based probes built on
these inhibitors will employ the same labeling mechanism
(Figure 1B).
Figure 2. Design and synthesis of ABP probes for monoamine oxi-
dases (MAO). A) Structures of sets of probes inspired by irreversible
monoamine oxidase inhibitors. B) Structures of irreversible MAO
inhibitors: pargyline (unspecific), deprenyl (specific for MAO B).
C) Synthesis of probe P1.
affect significantly the inhibition of either isozymes. Probe P3,
a racemic version of deprenyl equipped with an alkyne,
showed a decrease in inhibition of MAO B, whereas probe P1,
based on pargyline, showed an improvement of its IC50 value,
compared to that of the parent inhibitor.
Having shown that the designed probes meet the require-
ment of inhibition, we set out to study their use in vitro using
Pichia pastoris membrane preparations overexpressing
human monoamine oxidase. Recombinant human MAO
preparations were incubated with the corresponding probe
The straightforward synthesis of the ABPP probes started
with a reductive amination to introduce the desired prop-
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enzyme labeling by probe P1 (Figure 3E).
Collectively, these results demonstrate that
the developed ABPP system can serve as an
effective chemical tool for profiling activity
of both isoforms of MAO in vitro. These
valuable results prompted us to evaluate
further the potency and selectivity of the
best probes P1 and P3 in profiling the
activity of monoamine oxidases in far more
complex biological samples. Additionally,
we were interested in determining whether
these probes are able to target other flavin-
dependent enzymes.
First, we evaluated the general labeling
properties of probes P1 and P3 with differ-
ent mouse tissue homogenates (heart, lung,
brain). Interestingly, the only specific bands
were noticeable in the insoluble fraction of
mouse brain lysate (Figure S3 in the Sup-
porting Information). Probe P1, based on
the unspecific MAO inhibitor pargyline,
labeled two proteins in the range of
60 kDa, whereas probe P3, based on the
MAO B specific inhibitor deprenyl, labeled
only one target, which was identical to the
lower band labeled by probe P1. The label-
ing was dose-dependent and was still
observable at a concentration of 100 nm
(Figure S3B in the Supporting Informa-
tion). Interestingly and importantly, the
molecular weights of the labeled bands
matched up with those of MAO A (higher
band) and MAO B (lower band) (Fig-
ure S3C), and the labeling was completely
Figure 3. Fluorescent SDS-PAGE analysis of the labeling of monoamine oxidases A and B in
vitro. A) Screening of alkyne probes P1–P6 with MAO A and MAO B. B) Comparison of
fluorescence scanning (FS) and Coomassie Brilliant Blue (CBB) staining of the labeling of
MAO B by the probe P3. C) Labeling of MAO A and MAO B after heat denaturation (6 min
at 968C) (right) and without heat denaturation (left) of the enzyme. D) Concentration-
dependent labeling of MAO A and MAO B by probe P1. E) Competitive labeling of MAO A
and MAO B with pargyline and probe P1. F) Labeling of mixture of both MAO isoforms
with probes P1, P3, and P5.
for 1 h at room temperature followed by the attachment of
TAMRA-azide tag (Figure S2A in the Supporting Informa-
tion) by copper(I)-catalyzed alkyne–azide cycloaddition
(click chemistry, CC).[16] SDS-PAGE and in-gel fluorescence
scanning were employed to detect labeling events (Figure 3).
In these initial proof-of-concept experiments we could
demonstrate that both isoforms of MAO can be efficiently
labeled as a main target by probes P1, P3, and P5 (Figure 3A
and B) with slightly different isoform preferences (Figure 3F)
at concentrations as low as 100 nm (Figure 3D). Interestingly,
all potent probes identified in the screening (P1, P3, P5)
include methyl-substituted tertiary amines, indicating that
this feature might be important for efficient labeling. The
structural analogues without a methyl substituent at the
amino group (P2, P4, P6) exhibit very weak labeling,
suggesting that a secondary amino group decreases the
interactions between a probe and the protein or has
unfavorable redox behavior. Importantly, labeling is com-
pletely abolished when the protein is deactivated by heat
denaturation prior to incubation with the probe (Figure 3C),
suggesting that labeling occurs only with the active enzyme in
a specific manner. In competition experiments we could show
that our ABPP probes compete with MAO-specific inhibitors
for the same binding site (flavin cofactor) in the enzyme
active site since pargyline is able to efficiently block the
abolished when the lysate was first incubated with excess of
MAO inhibitors (Figure S3D).
Encouraged by these initial results, we rationalized that
since pargyline and deprenyl are applied in the study and
treatment of CNS disorders (deprenyl is used as an anti-
parkinson drug[32]), a human brain cancer cell line (designated
RAEW) isolated from a patient who was suffering from
a glioblastoma multiforme (GBM) tumor, would be a suitable
system for target validation.[34] Prior to ABPP labeling we
determined the cytotoxicity of probes P1 and P3 against an
eukaryotic cell line (GBM model, DBTRG-05MG) and
validated that within the range of concentrations used for
ABPP experiments the cells were still viable (Figure S4 in the
Supporting Information).
In situ labeling with GBM cells revealed that both probes
P1 and P3 labeled two bands in the range of 63 kDa as main
protein targets; however, the labeling by probe P3 proved to
be much more effective (Figure 4A). A 50 mm concentration
of probe P3 was sufficient for achieving binding saturation as
higher concentrations did not improve the labeling of the
much weaker lower band. Importantly, inhibitors of mono-
amine oxidases outcompeted the labeling by probe P3,
suggesting that these two proteins are MAO A (60.5 kDa)[35]
and MAO B (59.4 kDa).[35] For unequivocal identification of
the two probe-bound proteins we performed a quantitative
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studies indicating loss of MAO B specificity at high
concentrations.[37] However, P3 demonstrated higher
binding affinity towards MAO B (Figure 3F), which
was also noticeable in mouse brain tissue, in which
only the lower protein band, presumably MAO B, was
labeled (Figure S3A). Interestingly, in living human
brain tumor cells, this probe was bound preferentially
to MAO A. This important result can be explained by
the fact that the activity of MAO A is much higher in
intact cells than in the purified enzyme, which is known
to be particularly unstable at ambient temperatures
and loses its activity rapidly.[38] Moreover, one could
speculate that the topology of the mitochondrial outer
membrane (MOM) of both MAO isozymes can
influence the access of the probe to the enzyme.
Recent studies[39] demonstrated that MAO A is local-
ized at the cytosolic face in intact rat liver mitochon-
dria and in intact human placental mitochondria,[40]
while MAO B resides in the intermembrane space,[39]
which can pose some difficulties for the probe in MOM
permeability.[41] On the other hand, the weak labeling
of MAO B correlates with lower activity of MAO B
found in some cultured brain cells;[42] although both
MAO isoforms are expressed in human brain at similar
levels.[43] Taken together, these results underscore the
relevance of in situ studies on enzyme activity, which is
apparently not a simple function of enzyme abundance
but is tightly regulated by many dynamic processes
taking place exclusively in intact living cells.
Figure 4. A) In situ ABPP labeling of human brain cancer cells (insoluble
fraction, for the whole proteome see Figure S5 in the Supporting Information)
with probes P1 (lanes 5 and 6) and P3 (lanes 2–4). Competitive labeling with
MAO inhibitors deprenyl (Dep) and pargyline (Par) and probe P3 (lanes 7 and
8). B) Comparison of fluorescence scanning (FS) and Coomassie Brilliant Blue
(CBB) staining of labeling by the probe P3. C) Identification of lower band by
Western blotting (WB) using specific anti-MAO B antibodies.
In conclusion, we have presented the first activity-
based probes targeting a flavin-dependent oxidase. We
could demonstrate their utility in ABPP studies with
both tissues and live cells, particularly in exploring the
activity of monoamine oxidases. The unusual labeling
mechanism assured outstanding selectivity of the
probe molecules which made it possible to study the
potential off-target interactions of the clinically used drug
deprenyl. We could show that it reacted exclusively with
MAO A and B. However, we are convinced that the scope of
our novel chemoproteomic approach can be extended by
careful fine-tuning of the probe core structure which can
result either in higher specificity of the probes or a broader
spectrum of the targeted flavin oxidases. Research in this
direction is currently underway in our laboratories.
proteomic analysis using a trifunctional reporter tag (biotin-
TAMRA-azide, Figure S2B), which was attached to a probe
P3 under CC conditions to allow visualization, enrichment,
and subsequent identification of proteins by mass spectrome-
try.[4e] Analysis of peptide fragments employing the
SEQUEST algorithm identified MAO A with nearly 40%
protein coverage (upper band on a fluorescent SDS-PAGE).
Unfortunately, the enrichment on avidin beads was insuffi-
cient for the lower protein band; however, this protein was
unambiguously identified as MAO B by Western blot analysis
using specific anti-MAO B antibodies (Figure 4C).
Received: March 12, 2012
Published online: && &&, &&&&
The results of labeling in live brain cells strikingly
demonstrate that the covalently binding inhibitors pargyline
Keywords: activity-based probes · deprenyl · flavin cofactor ·
monoamine oxidases · proteomics
.
and deprenyl act very selectively with MAO A and B but with
no other protein targets. This outstanding selectivity is
triggered by a unique “suicide” inhibition mechanism that is
customized for this enzyme family. This is in contrast to other
ABPP studies which identified many off-targets of clinically
used covalent drugs.[36]
Probe P3, based on the MAO B specific inhibitor
deprenyl, showed distinct isozyme preference in in vitro and
in situ labeling. In experiments with recombinant proteins,
this probe labeled both isoforms of MAO in agreement with
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www.angewandte.org
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6
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Proteomics
High profile: New activity-based protein
profiling (ABPP) probes have been
designed that target exclusively mono-
amine oxidases A and B within living cells
(see picture; FAD=flavin adenine dinu-
cleotide, FMN=flavin monodinucleo-
tide). With these probes it could be
shown that the MAO inhibitor deprenyl,
which is in clinical use against Parkin-
son’s disease, shows unique protein
specificity despite its covalent mecha-
nism of action.
J. M. Krysiak, J. Kreuzer, P. Macheroux,
A. Hermetter, S. A. Sieber,*
R. Breinbauer*
&&&& — &&&&
Activity-Based Probes for Studying the
Activity of Flavin-Dependent Oxidases
and for the Protein Target Profiling of
Monoamine Oxidase Inhibitors
Angew. Chem. Int. Ed. 2012, 51, 1 – 7
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!