A Fluorescent-Labeled Phosphono Bisbenzguanidine As an Activity-Based Probe for Matriptase

Article abstract of DOI:10.1002/chem.201700319

Activity-based probes are compounds that exclusively form covalent bonds with active enzymes. They can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. The design of a new activity-based probe for matriptase, a member of the type II transmembrane serine proteases, is based on linker-connected bis-benzguanidines. An amino acid, introduced as linker, bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine. The resulting irreversible mode of action was demonstrated, leading to enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase. The ten-step synthetic approach to a coumarin-labeled bis-benzguanidine and its evaluation as activity-based probe for matriptase based on in-gel fluorescence and fluorescence HPLC is reported. HPLC fluorescence detection as a new application for activity-based probes for proteases is demonstrated herein for the first time.

Full text of DOI:10.1002/chem.201700319

DOI: 10.1002/chem.201700319
Communication
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Protease Probes
A Fluorescent-Labeled Phosphono Bisbenzguanidine As an
Activity-Based Probe for Matriptase
Daniela Hꢀußler,[a] Anna-Christina Schulz-Fincke,[a] Anna-Madeleine Beckmann,[a]
Aline Keils,[b] Erik Gilberg,[a, c] Martin Mangold,[a] Jꢁrgen Bajorath,[c] Marit Stirnberg,[a]
Torsten Steinmetzer,[b] and Michael Gꢁtschow*[a]
triad, serine, histidine and aspartate, essential for proteolytic
activity.[1] Matriptase, one of the best characterized TTSPs, is
Abstract: Activity-based probes are compounds that ex-
clusively form covalent bonds with active enzymes. They
can be utilized to profile enzyme activities in vivo, to iden-
tify target enzymes and to characterize their function. The
design of a new activity-based probe for matriptase,
a member of the type II transmembrane serine proteases,
is based on linker-connected bis-benzguanidines. An
amino acid, introduced as linker, bears the coumarin fluo-
rophore. Moreover, an incorporated phosphonate allows
for a covalent interaction with the active-site serine. The
resulting irreversible mode of action was demonstrated,
leading to enzyme inactivation and, simultaneously, to
a fluorescence labeling of matriptase. The ten-step syn-
thetic approach to a coumarin-labeled bis-benzguanidine
and its evaluation as activity-based probe for matriptase
based on in-gel fluorescence and fluorescence HPLC is re-
ported. HPLC fluorescence detection as a new application
for activity-based probes for proteases is demonstrated
herein for the first time.
mainly expressed in epithelia, such as epidermis or thymic
stoma.[2–4] The proteolytic activity of matriptase is regulated by
the Kunitz type hepatocyte growth factor activator inhibitors
HAI-1 and HAI-2.[5,6] Matriptase is expressed as inactive zymo-
gen precursor and has to be converted in an autocatalytic
manner into its active form.[7]
Deregulation of matriptase is related to a variety of epithelial
cancers and enhanced metastasis; increased matriptase activi-
ties often correlate with a poor disease outcome. Matriptase
processes and activates several substrates, which themselves
play critical roles in tumorigenesis, such as hepatocyte growth
factor/scatter factor, urokinase-type plasminogen activator and
protease activated receptor 2.[8–10]
Furthermore, matriptase, as an inducer and activator of pro-
collagenases, was found to be a key initiator of cartilage de-
struction in osteoarthritis. It is able to activate selective pro-
matrix metalloproteinases and to induce collagenase expres-
sion.[11] Several intestinal diseases are linked to a reduced ma-
triptase activity and to an impaired intestinal barrier func-
tion.[12,13] Recent findings propose that matriptase activates he-
magglutinin of certain H9N2 and H1N1 influenza A viruses and
promotes viral replication. Hence, inhibition of matriptase sig-
nificantly blocked influenza virus replication.[14–16]
Matriptase, the eponymous enzyme of the matriptase subfami-
ly, is a member of the type II transmembrane serine proteases
(TTSPs), a family of mammalian cell surface-associated serine
proteases with a unique modular structure. These membrane-
anchored proteases are structurally defined by a cytoplasmic
N-terminal tail, a transmembrane domain, a stem region that
contains various functional domains, and a C-terminal extracel-
lular serine protease domain, characterized by the catalytic
Activity-based probes (ABPs) have emerged as a powerful
tool in protein identification and profiling. The probes’ ability
to selectively visualize only the active forms of proteases is ad-
vantageous because expression levels often do not correlate
with their enzymatic activity, for example, due to post-transla-
tional regulation.[17,18] Typically, the probes consist of three es-
sential components, that is, a reactive group for covalent inter-
action, a recognition element that controls the probe’s selec-
tivity and a detectable agent. Several radioactive, fluorescent
and biotin labels are established reporter tags for ABPs.[17,18]
While fluorescent ABPs for matriptase have not been reported
so far, a biotinylated chloromethyl ketone peptide has been
used for the detection of active matriptase.[19] Moreover,
biotin-labeled ABPs for trypsin-like enzymes with a phospho-
nate warhead and one benzamidine moiety have been devel-
oped.[20]
[a] Dr. D. Hꢀußler, A.-C. Schulz-Fincke, Dr. A.-M. Beckmann, E. Gilberg,
M. Mangold, Dr. M. Stirnberg, Prof. Dr. M. Gꢁtschow
Pharmaceutical Institute, Pharmaceutical Chemistry I
University of Bonn, An der Immenburg 4
53121 Bonn (Germany)
E-mail: guetschow@uni-bonn.de
[b] A. Keils, Prof. Dr. T. Steinmetzer
Institute of Pharmaceutical Chemistry
Philipps University of Marburg, Marbacher Weg 6
35032 Marburg (Germany)
[c] E. Gilberg, Prof. Dr. J. Bajorath
Department of Life Science Informatics
B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry
University of Bonn, Dahlmannstr. 2, 53113 Bonn (Germany)
In continuation of a previous study,[24] our design of an ABP
for matriptase is based on two linker-connected benzguani-
dines, a known substructure of matriptase inhibitors.[21–23] One
benzguanidine moiety is very likely to be oriented towards the
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/chem.201700319.
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deep, negatively charged S1 pocket.[25,26] However, the remain-
ing benzguanidine is either able to address the S3/S4 or the S2
pocket of matriptase.[4,27–29] The assembly of our probe was in-
spired by several potent dibasic matriptase inhibitors, such as
peptidic ketobenzothiazoles derived from the natural Arg-Gln-
Ala-Arg autoactivation sequence of matriptase,[27,28] bis-benza-
midines,[4,29] or sulfonylated 3-amidinophenylalanine deriva-
tives.[30] To achieve covalent interaction, a phosphonate group
was attached. The a-aminoalkylphosphonates represent phos-
phonic analogues of naturally occurring amino acids which can
be further extended by a suitable peptide moiety to improve
target selectivity. Such peptidic phosphonates are well estab-
lished irreversible inhibitors of serine proteases.[31–39] The at-
tachment of a coumarin fluorophore was accomplished by the
introduction of the trifunctional amino acid lysine. Coumarins
represent a widely used class of fluorescent dyes, distinguished
by their small molecular size, low bleaching and large Stokes
shifts.[40–44]
pound 10 was then coupled with the before produced amino-
phosphonate 5.[36,38] This led to 11, a dipeptide intermediate
containing a coumarin tag and the phosphonate group. The
ammonium salt 12, obtained by deprotection of 11, was con-
verted to the free amine and reacted with 4-nitrobenzoic acid
(13) in order to incorporate a second nitro moiety. A HATU-
promoted coupling yielded the bis-nitro compound 14. The re-
duction of the nitro groups was carried out with SnCl2.[33,39] Fol-
lowing a literature protocol,[33,37] the amino groups of 15 were
converted to protected guanidine moieties in intermediate 17.
For this purpose, a HgCl2-mediated reaction with N,N’-di-Boc-S-
methylisothiourea (16) was applied. After the removal of the
Boc-protecting groups with trifluoroacetic acid, the desired
bis-benzguanidine 18 was received and purified by preparative
HPLC. The hydrochloride salt was obtained by adding HCl to
an aqueous solution of the guanidinium trifluoroacetate, fol-
lowed by lyophilisation.
The ABP 18 was characterized for its spectroscopic proper-
ties in buffer and exhibited an absorption maximum of
369 nm, an emission maximum of 448 nm, and thus a Stokes
shift of 79 nm (Figure S1 in the Supporting Information).
The inhibitory potency of the ABP 18 against matriptase was
determined with the fluorogenic substrate Mes-d-Arg-Pro-Arg-
AMC. The probe showed time-dependent inhibition (Figure 1),
and non-linear regression of the progress curves gave pseudo-
first order rate constants of irreversible inhibition, kobs. These
values were plotted versus the inhibitor concentrations
(Figure 1, right inset) and non-linear regression gave kinac/Ki.
This value, which characterizes the ability of a covalent irrever-
sible inhibitor to interact with a target was 576 mÀ1 sÀ1. ABP 18
was also evaluated at related serine proteases. At a concentra-
tion of 30 mm, it did not inhibit human thrombin and bovine
Scheme 1 outlines the convergent synthetic approach to the
ABP 18, similar to the one that has previously been established
for other benzguanidino phosphonates.[24] Details are given in
the Supporting Information. The racemic Cbz-protected a-ami-
nophosphonate 4 was prepared by a three-component reac-
tion, comprising triphenyl phosphite (1), benzyl carbamate (2)
and 4-nitrobenzaldehyde (3).[32–34] Deprotection of the amino-
phosphonate 4 was accomplished with a solution of HBr in
acetic acid.[36–38] The bromide salt 5 was used in the next step
without further purification. To produce the coumarin-labeled
amino acid 10, a Knoevenagel reaction was performed with 2-
hydroxy-4,5-dimethoxybenzaldehyde (6) and Meldrum’s acid
(7),[41] and the resulting 6,7-dimethoxy-coumarin-3-carboxylic
acid (8) was reacted with Na-Boc-protected lysine (9).[44] Com-
Scheme 1. Synthesis of compound 18. Reaction conditions: a) AcOH/HBr, RT; b) HATU, DIPEA, DMF, RT, 38% yield from 4 to 11; c) TFA, CH2Cl2, RT; d) HATU,
DIPEA, DMF, RT, 74% yield from 11 to 14; e) SnCl2·2H2O, H2O, EtOAc, reflux, 15% yield; f) HgCl2, NEt3, CH2Cl2, RT; g) TFA, CH2Cl2, RT, prep. HPLC, 21% yield
from 15 to 18. DIPEA=N,N-Diisopropylethylamine, TFA=trifluoroacetic acid.
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Figure 1. Inhibition of human matriptase by activity-based probe 18. Black:
uninhibited reaction; magenta: 4 mm; green: 8 mm; orange: 12 mm; purple:
16 mm; blue: 20 mm. Values kobs were obtained from non-linear regression of
the progress curves using the equation [P]=vi (1ÀeÀkobs·t)/kobs +d, where [P]
is the product concentration, vi is the initial rate and d is the offset. The left
inset shows the first 800 s of the progress curves. The right inset shows
a plot of kobs values (mean of duplicate measurements) versus the inhibitor
concentrations, [I]. For non-linear regression, the following equation was
used kobs =kinac·[I]/(Ki (1+[S]/Km)+[I]), where kinac is the first-order inactivation
rate constant, Ki is the inhibition constant and [S] is the substrate concentra-
tion. A kinac/Ki value of 576Æ151mÀ1 sÀ1 was obtained. The standard error
refers to the non-linear regression of the kobs versus [I] plot.
Figure 2. Modeled covalent complex of 18 and matriptase.
free to move. The calculations yielded a plausible binding
mode as shown in Figure 2. In accordance with previous re-
ports,[45–47] one of the (partially) negatively charged oxygen
atoms of the phosphonate is proposed to form ionic interac-
tions with the protonated e2-nitrogen of His57, whereas the
other is orientated towards the oxyanion hole. The “right”
benzguanidinium moiety, as an arginine mimetic, expectedly
interacts with Asp189 in the deep S1 pocket by charge-assisted
hydrogen bonding. Possible interactions in the S2 and S3/S4
pockets were modeled with lower confidence. In the putative
binding mode depicted in Figure 2, the “left” benzguanidinium
group occupies the upper S2 pocket forming putative hydro-
gen bonds with the carbonyl oxygen atoms of His57 and
Cys58, and the coumarin moiety is oriented towards the S3/S4
factor Xa, where more than 30% product formation was ob-
served after 30 min. However, the closest relative of matriptase,
that is, matriptase-2, was also inhibited, although to a lesser
extent (kinac/Ki ÆSEM=79.3Æ3.6mÀ1
s
À1). An applicability of the
probe to label matriptase should nevertheless be possible, be-
cause, in comparison to matriptase, the liver enzyme matrip-
tase-2 has a limited tissue distribution.
The initial parts of the progress curves of the matriptase-cat-
alyzed reaction in the presence of 18 were nearly linear
(Figure 1, left inset), indicating a slow irreversible inhibition at
the beginning of the measurement. We assume a rapid equilib-
rium between the free enzyme and two reversible enzyme-in-
hibitor complexes, from which one is non-productive with re-
spect to the inactivation step. In such a complex, the “left”
benzguanidine moiety might occupy the S1 pocket. However,
when the “right” benzguanidine moiety binds to the S1
pocket, an inactivation would be facilitated because the phos-
phonate warhead would be directed to the active site serine
residue.
In order to propose possible binding modes of probe 18,
molecular docking calculations were performed, using the crys-
tal structure of a complex of matriptase with a bis-benzami-
dine inhibitor (PDB-ID: 4JZI).[4] The interaction of serine pro-
teases with diphenyl phosphonate inhibitors includes the for-
mation of a serine phosphono diester through the nucleophilic
attack of the active site residue Ser195 at the phosphorus and
the release of one phenoxy group, followed by a hydrolytic
“aging” into a phosphono monoester.[45–47] Such a monoester
complex with phosphorus in a tetrahedral configuration was
generated by covalent docking as follows. Both phenoxy
groups were removed from the inhibitor and the single bond
between the oxygen of Ser195 and the phosphorus was man-
ually built. In the presence of this covalent constraint, the in-
hibitor was flexibly docked and the Ser195 residue was also
Figure 3. Imaging of human matriptase with the fluorescent probe 18. Dif-
ferent amounts of matriptase (1–5 mg) were incubated with 50 mm of 18. La-
beling reactions were performed for 45 min at 378C. The proteins were sep-
arated by SDS–PAGE and visualized by (A) fluorescence detection and
(B) Coomassie staining. Matriptase (3 mg), matriptase (3 mg) spiked with
human embryonic kidney (HEK) lysate (25 mg), and HEK lysate (25 mg) alone
were incubated for 45 min at 378C with 50 mm of 18. The proteins were sep-
arated by SDS–PAGE and visualized by (C) fluorescence detection and
(D) Coomassie staining. M, molecular mass marker.
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Figure 4. Size-exclusion chromatograms of human matriptase labeled with the fluorescent probe 18. The fluorescence detector gain was adjusted to 10.
(A) Matriptase (5 mg) was incubated for 15 min (blue), 30 min (magenta) or 45 min (black) at 378C with 50 mm of 18. The protein was subjected to HPLC and
visualized by fluorescence detection (lex =369 nm, lem =448 nm). (B–D) Proteins were separated by size-exclusion chromatography and simultaneously visual-
ized by UV (labs =280 nm) and fluorescence detection (lex =369 nm, lem = 448 nm). Blue: UV channel; black: fluorescence channel. (B) Matriptase (3 mg), (C)
HEK lysate (25 mg) spiked with matriptase (3 mg), and (D) HEK lysate (25 mg) alone were incubated for 45 min at 378C with 50 mm of 18.
region, but does not engage in well-defined ligand–protein in-
teractions.
tained with matriptase in the presence of a large excess of
lysate proteins (Figure 4C) clearly demonstrated the suitability
and selectivity of ABP 18.
The feasibility of ABP 18 for direct in-gel fluorescence detec-
tion of matriptase was demonstrated. In addition, we have ex-
amined whether matriptase could be visualized after labeling
with 18 and passage through an HPLC system equipped with
a fluorescence detector. Different amounts of matriptase (1–
5 mg) were treated with 50 mm of 18 for 45 min. After perform-
ing the SDS–PAGE (Figure 3A), a band at 26 kDa could be de-
tected and its intensity correlated well with the increasing
amount of matriptase. At the lowest protease amount of 1 mg,
detection was still possible. The presence of matriptase was
confirmed by Coomassie Brilliant Blue staining (Figure 3B).
The mixture of matriptase (5 mg) and 18 (50 mm) was sub-
jected to size-exclusion chromatography after different incuba-
tion times of 15, 30, and 45 min and analyzed by means of
fluorescence measurement. We obtained a clear single peak
for the enzyme–probe complex. The signal increased depend-
ing on the incubation time, reflecting the successive increase
of the complex concentration (Figure 4A). When lower
amounts of matriptase (1 or 3 mg) were treated with 18
(50 mm), the observed intensities of the signals were less dis-
tinct after incubation times of 15, 30, and 45 min (Figure S2 in
the Supporting Information). Even an amount of only 1 mg of
matriptase led to a chromatogram with an excellent signal-to-
noise ratio.
By applying the chemotype of phosphono bis-benzguani-
dines, the first fluorescent ABP of matriptase was successfully
employed for direct in-gel fluorescence readout and HPLC size-
exclusion chromatography coupled to fluorescence detection.
The probe described herein is expected to serve as a valuable
tool compound for future investigations of matriptase, an
enzyme of strong therapeutic importance.
Acknowledgements
D.H. was supported by the Bonn International Graduate School
of Drug Sciences (BIGS DrugS) and the NRW International
Graduate Research School Biotech-Pharma, A.M.B. and M.S. by
the German Research Foundation (DFG) Grant STI 660/1-1.
Keywords: activity-based probes · fluorescence · matriptase ·
peptidomimetics · phosphonates
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Manuscript received: January 22, 2017
Revised: February 24, 2017
Final Article published: && &&, 0000
Chem. Eur. J. 2017, 23, 1 – 6
www.chemeurj.org
5
ꢂ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Communication
COMMUNICATION
&
Protease Probes
D. Hꢀußler, A.-C. Schulz-Fincke,
A.-M. Beckmann, A. Keils, E. Gilberg,
M. Mangold, J. Bajorath, M. Stirnberg,
T. Steinmetzer, M. Gꢁtschow*
&& – &&
A Fluorescent-Labeled Phosphono
Bisbenzguanidine As an Activity-Based
Probe for Matriptase
An activity-based probe for matriptase,
a therapeutically important transmem-
brane serine protease, was designed,
synthesized and simultaneously evaluat-
ed by in-gel fluorescence analysis and
HPLC fluorescence detection.
&
&
Chem. Eur. J. 2017, 23, 1 – 6
www.chemeurj.org
6
ꢂ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!