Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Article abstract of DOI:10.1016/j.bmcl.2016.11.064

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elast

Full text of DOI:10.1016/j.bmcl.2016.11.064

Accepted Manuscript
Fluorescent diphenylphosphonate-based probes for detection of serine protease
activity during inflammation
Laura E. Edgington-Mitchell, Nicholas Barlow, Luigi Aurelio, Aminath Samha,
Monika Szabo, Bim Graham, Nigel Bunnett
PII:
S0960-894X(16)31218-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.064
BMCL 24459
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 September 2016
21 November 2016
22 November 2016
Please cite this article as: Edgington-Mitchell, L.E., Barlow, N., Aurelio, L., Samha, A., Szabo, M., Graham, B.,
Bunnett, N., Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during
inflammation, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.11.064
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Fluorescent diphenylphosphonate-based probes for detection of serine
protease activity during inflammation
Laura E. Edgington-Mitchell1*, Nicholas Barlowb, Luigi Aureliob, Aminath Samhaa,
Monika Szabob, Bim Grahamb* & Nigel Bunnetta,c,d*
a Drug Discovery Biology, b Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
c Department of Pharmacology and Therapeutics University of Melbourne,
Parkville, VIC, Australia
d Departments of Surgery and Pharmacology, Columbia University College of
Physicians and Surgeons, New York, NY, USA (present address)
*Correspondence should be addressed to:
Laura E. Edgington-Mitchell, email: laura.edgington-mitchell@monash.edu
Nigel Bunnett, email: nigel.bunnett@monash.edu
Bim Graham, email: bim.graham@monash.edu
KEYWORDS: fluorescent probes, activity-based probes, trypsin, elastase,
protease, pancreatitis, colitis, inflammation, protease inhibitors,
diphenylphosphonate
ABBREVIATIONS: ABP (activity-based probe); DPP (diphenylphosphonate); NE
(neutrophil elastase); PR-3 (proteinase 3)
ABSTRACT
Activity-based probes are small molecules that covalently bind to the active site
of a protease in an activity-dependent manner. We synthesized and
characterized two fluorescent activity-based probes that target serine proteases
with trypsin-like or elastase-like activity. We assessed the selectivity and potency
of these probes against recombinant enzymes and demonstrated that while they
are efficacious at labeling active proteases in complex protein mixtures in vitro,
they are less valuable for in vivo studies. We used these probes to evaluate
serine protease activity in two mouse models of acute inflammation, including
pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was
increased during pancreatitis. Levels of elastase-like proteases were low in
pancreatic lysates and colonic luminal fluids, whether healthy or inflamed.
Exogenously added recombinant neutrophil elastase was inhibited upon
incubation with these samples, an effect that was augmented in inflamed
samples compared to controls. These data suggest that endogenous inhibitors
and elastase-degrading proteases are upregulated during inflammation.
1

Proteases are enzymes that cleave proteins to mediate critical processes during
normal physiology and disease. Covalent activity-based probes (ABPs) are
unique tools that can measure the activity of specific proteases within complex
protein mixtures1-4. Probes incorporating biotin or click chemistry groups can be
used for biochemical assessment of activity and for identification of probe targets
after affinity purification. Fluorescent ABPs allow for shorter biochemical assays,
as labeling can be detected directly in the gel without the need for click reactions,
membrane transfer or streptavidin binding. Furthermore, fluorescent ABPs can
be used to visualize protease activity in situ, by whole animal or tissue imaging or
fluorescent microscopy. Fluorescent ABPs for cysteine proteases have been well
characterized and are efficacious in vivo5-13. Despite the critical importance of
serine proteases in physiological and pathophysiological processes, ABPs for
these enzymes are scarce, and they have not been extensively characterized in
complex biological fluids or in animal models14-22. We therefore aimed to
synthesize and characterize fluorescent ABPs that can detect serine proteases
that are activated during inflammation, namely trypsin- and elastase-like
proteases23-28
.
There are few electrophilic warheads that react with serine proteases. This is
largely owing to the fact that the hydroxyl group in the active site is not as
nucleophilic as the thiol group of cysteine proteases. Most of the reported
warheads react broadly with serine hydrolases (fluorophosphonates29, β-
lactams30, β-lactones31), and some also bind to lipases (sulfonyl fluorides32,33).
Phosphonates14,17, phosphoramidates21, and 4-chloroisocoumarins19,20,22 are
more specific for serine proteases. For our fluorescent ABPs, we elected to use
a diphenylphosphonate (DPP) moiety based on its reported stability in aqueous
environments and the irreversible nature of its binding to the serine protease
hydroxyl group14,16,17,34. Whether fluorescent DPP probes are suitable for in vivo
use has not previously been reported.
We based our design on two existing biotin probes with DPP warheads. Biotin-
Pro-Lys-DPP targeted trypsin-like proteases, including trypsin, tryptase, plasmin
and thrombin17. Biotin-Val-DPP14 was reported to label elastase-like proteases.
In both cases, we replaced the biotin tag and PEG spacer with a Cy5 tag to
generate Cy5-PK-DPP and Cy5-V-DPP (Scheme 1). Cy5 is a near-infrared
fluorophore that is well suited for in vivo imaging7,8.
For
the
synthesis
of
Cy5-PK-DPP
(Scheme
1),
diphenyl
was
benzyloxycarbonylamino-(4-phthalimidobutyl)methanephosphonate
prepared from 5-amino-1-pentanol by phthalamide protection, Swern oxidation
and Birum-Oleksyszyn condensation, following the procedures described by
Joossens et al35. Benzylcarbamate (Z) deprotection by hydrogenation was
followed by amide coupling to Z-protected proline in the presence of HOBt and
EDCI, providing a Z-protected intermediate that could be purified by silica gel
chromatography. Deprotection of the Z group, again by hydrogenation, followed
by HCTU-mediated amide coupling to sulfonated Cy5 carboxylic acid provided a
2

Scheme 1. Synthesis of probes. Reagents and conditions: (a) 10% Pd/C, H2, MeOH, 2-6 h; (b) Cbz-
Pro-OH, HOBt, EDCl, Et3N, MeCN, 15 h; (c) sulfonated Cy5 carboxylic acid, HCTU or PyBOP, Et3N,
5 h-overnight; (d) 30% N2H4.H2O, MeCN/H2O, 15 h.
fluorescent intermediate, which was purified by preparative HPLC. The
phthalamide group was then removed by treatment with hydrazine to provide
Cy5-PK-DPP, which was isolated in pure form following further preparative HPLC
(Fig S1).
An analogous synthetic approach was applied for the preparation of Cy5-V-DPP
(Scheme 1). The Birium-Oleksyszyn condensation was performed with
isobutylaldehyde to provide diphenyl benzyloxycarbonylaminoisopropyl-
methanephosphonate. Deprotection of the Z group was performed by
hydrogenation, and the crude amine was coupled to sulfonated Cy5 carboxylic
acid in the presence of PyBOP, providing pure Cy5-V-DPP after preparative
HPLC (Fig S2).
We first tested
the ability of the
fluorescent
ABPs to label
key
proteases. The
probes were
target
incubated with
recombinant
proteases,
Figure 1. Concentration-dependent labeling of recombinant serine
proteases. A) Recombinant trypsin, elastase, and proteinase-3 were incubated
with increasing doses of Cy5-PK-DPP or Cy5-V-DPP. Proteins were resolved by
SDS-PAGE and in-gel fluorescence was measured to detect protease labeling.
B) Protease labeling in A was quantified using densitometry (n=3).
followed
by
fluorescent
SDS-PAGE
analysis. Cy5-
3

PK-DPP labeled recombinant trypsin in a concentration-dependent manner (Fig
1A,B). Cy5-V-DPP labeled recombinant neutrophil elastase (NE) and proteinase
3 (PR-3).
Next, the selectivity of the probes
against a panel of recombinant
serine and cysteine proteases was
examined (Fig 2). Equal amounts of
each recombinant protease were
labeled with 1 µM probe, followed by
fluorescent SDS-PAGE analysis.
Cy5-PK-DPP
trypsin, thrombin and plasmin, but
was unreactive towards
strongly
targeted
chymotrypsin, NE, and PR-3. This is
consistent with the specificity of the
biotin-tagged version of this probe17.
Like Biotin-PEG-succinyl-V-DPP14,
Cy5-V-DPP strongly labeled NE. It
also labeled PR-3, albeit to a lesser
extent. Very low interaction with
trypsin, chymotrypsin, and thrombin
was observed. As the DPP warhead
is selective for serine proteases,
neither of the probes reacted with
the cysteine protease cathepsin S.
Figure 2. Selectivity of activity-based probes. Cy5-
PK-DPP or Cy5-V-DPP incubated with a panel of
recombinant serine proteases (elastase, proteinase-3,
trypsin, chymotrypsin, thrombin, plasmin, and
granzyme B) and one cysteine protease (cathepsin S).
Proteins were resolved by SDS-PAGE and protease
labeling was detected by measuring in-gel
fluorescence.
Having demonstrated that these
probes can detect the activity of
purified serine proteases, we then
examined their efficacy in complex
protein mixtures (Fig 3A). Bone marrow cells were extracted from healthy mice,
lysed, and incubated with the probes. Cy5-PK-DPP labeling was observed at
both 1 and 10 µM. Cy5-V-DPP labeled one protease, but only at 10 µM. In an
immunoprecipitation assay with a NE-specific antibody, we confirmed that this
band was NE (Fig 3B). Cy5-V-DPP also labeled a high molecular weight species
that did not immunopreciptate with the NE antibody (Fig 3A-B). Based on its size,
this may be a serum protein that binds to the probe non-specifically.
Next, we analyzed serine protease activity in a mouse model of pancreatitis in
which multiple proteases become activated. Pancreatic lysates were prepared
from normal mice and those with acute pancreatitis induced by the secretogogue
caerulein. As expected, Cy5-PK-DPP exhibited increased labeling of trypsin-like
proteases in the 25-KDa region in the inflamed
4

pancreas (Fig 4A). As in the bone
marrow lysate, a high molecular
weight species was detected with the
Cy5-V-DPP probe, though no NE
labeling was observed.
To confirm that Cy5-V-DPP is capable
of detecting NE activity in pancreatic
lysates, we spiked recombinant NE
(rNE) into pancreas lysates from
normal and inflamed mice. The spiked
rNE was successfully detected by
Cy5-V-DPP. Compared to samples
containing rNE alone, however, the
labeling in the spiked samples was
greatly reduced (Fig 4B). The
reduction of rNE activity was greater in
inflamed pancreas lysates than in the
Figure 3. ABP labeling in complex protein
mixtures. A) Bone marrow cells were isolated,
lysed, and incubated with Cy5-PK-DPP or Cy5-V-
DPP at 1 or 10 µM. Proteins were resolved by
SDS-PAGE and protease labeling was detected
by
measuring
in-gel
of
fluorescence.
Cy5-V-DPP-labeled
B)
Immunoprecipitation
lysates in A with an elastase-specific antibody,
followed by SDS-PAGE and in-gel fluorescence
detection.
normal
pancreas
samples, though
this
did
not
reach
significance due
to
sample
variability
(p=0.09). These
results suggest
that
activity
elastase
is
somehow
suppressed in
pancreatic
lysates, and that
these
may
effects
be
increased
during
Figure 4. Ex vivo labeling of protease activation during acute
pancreatitis. A) Lysates were prepared from normal and inflamed pancreas
and incubated with Cy5-PK-DPP or Cy5-V-DPP (10 µM). Proteins were
resolved by SDS-PAGE and protease labeling was detected by in-gel
fluorescence. B) Recombinant neutrophil elastase was incubated with Cy5-V-
DPP (1 µM), either alone (rNE) or in the presence of pancreas lysates
(normal/inflamed + rNE). Elastase activity was detected by in-gel fluorescence
(top/red). Total elastase levels were then measured by western blot
(bottom/blue). Bands were quantified by densitometry. (Activity: rNE v Norm
p=4.1e-5; rNE v Infl p=1.4e-5; Norm v Infl p=0.09. Total: rNE v Norm
p=0.0005; rNE v Infl p=0.008; Norm v Infl p=0.97. Norm Act v Tot p=0.0003;
Infl Act v Tot p=0.0006).
pancreatitis. To
investigate this
further,
examined total
rNE levels in the
reactions
Western
we
by
blot.
The rNE that
5

was spiked into
pancreatic lysates
was less abundant
despite the initial
addition of equal
amounts.
Furthermore,
the
higher molecular-
weight forms of
elastase were no
longer
present,
suggesting
degradation
by
pancreatic
proteases.
activity
The
was
significantly lower
than the total
amount of elastase
present (when
normalized as
a
percentage or rNE
alone), suggesting
that
inhibitors contribute
to the reduced
endogenous
activity in addition
degradation.
Figure 5. Ex vivo labeling of protease activation during acute colitis. A)
Luminal fluids were collected from normal and inflamed colons and
incubated with Cy5-PK-DPP or Cy5-V-DPP (1 µM). Proteins were resolved
by SDS-PAGE and protease labeling was detected by measuring in-gel
fluorescence. B) Recombinant neutrophil elastase was incubated with Cy5-
V-DPP, either alone (rNE) or in the presence of luminal fluids
(normal/inflamed + rNE). Elastase activity was then detected by in-gel
fluorescence (top/red) followed by western blotting for total elastase
(bottom/blue) Bands were quantified by densitometry. (Activity: rNE v Norm
p=0.0001; rNE v Infl p=1.7e-5; Norm v Infl p=0.03. Total: rNE v Norm
p=0.15; rNE v Infl p=0.05; Norm v Infl p=0.96. Norm Act v Tot p=0.008; Infl
Act v Tot p=0.0001). C) Samples were treated as in B except rNE was
preinbuated with the samples for 10 minutes prior to Cy5-V-DPP addition.
(Activity: rNE v Norm p=0.006; rNE v Infl p=0.005; Norm v Infl p=0.16. Total:
rNE v Norm p=0.05; rNE v Infl p=0.005; Norm v Infl p=0.43. Norm Act v Tot
p=0.13; Infl Act v Tot p=0.002). D) Measurement of NE activity in luminal
fluids of normal and inflamed mice in the presence and absence of rNE
using the fluorogenic substrate probe AAPV-AMC (n=3 mice per condition).
We also examined
serine
protease
activity in a mouse
model of acute
colitis (Fig 5A).
Using
Cy5-PK-
DPP, we observed
strong labeling of
two
trypsin-like
proteases in luminal
colon fluids. There
was considerable
variation in the
activation of these proteases in both the normal and inflamed groups and thus no
difference between the two groups. Minimal labeling was observed with the Cy5-
V-DPP probe. As we observed in the pancreas lysates, rNE lost activity upon
incubation with luminal fluids of normal mice, and this effect was significantly
6

exacerbated in fluids from mice with colitis (Fig 5B). Western blotting for elastase
revealed a reduction in total rNE levels upon incubation with luminal fluids. This
effect was more apparent when rNE was pre-incubated with the luminal fluids for
10 minutes prior to Cy5-V-DPP addition (Fig 5C). The reduction in activity was
also confirmed using the AAPV-AMC fluorogenic substrate probe, an
independent measure of elastase activity (Fig 5D). These data suggest the
presence of elastase-degrading proteases as well as endogenous inhibitors in
luminal fluids. Thus, elastase degrading/inhibiting factors are likely to be
upregulated in diverse inflammatory settings. Serpin B1, elafin, and secretory
leukocyte protease inhibitor (SLPI) are endogenous inhibitors of elastase. All
three inhibitors were reported to be increased in colonic biopsies from patients
with ulcerative colitis, which supports our hypothesis.28,36 Trypsin-like proteases
in both pancreatic lysates and luminal fluids are likely responsible for elastase
degradation.
It is possible that tissue lysis resulted in a disruption of the compartmentalization
of elastase, its inhibitors and degrading proteases in both of these models. For
this reason, it is most preferable to analyze protease activity in its native
environment without manipulation of the tissue. Thus, we aimed to monitor
serine protease activity in vivo. We administered the probes to mice with
Figure 6. In vivo imaging of pancreatitis with ABPs. A) Healthy mice or those with acute pancreatitis
were injected with Cy5-PK-DPP, Cy5-V-DPP, or vehicle control (No Probe). After 5 hours, pancreata
were removed and imaged for Cy5 fluorescence. Gain settings are indicated to the right of each image
series. B) Quantification of absolute levels of Cy5 fluorescence measured in A with background
subtracted (no-probe control). C) Biochemical analysis of protease labeling in the tissues from A.
Pancreata were lysed and proteins were resolved by SDS-PAGE. Protease labeling was detected by
measuring in-gel fluorescence. Note autofluorescent background bands in the no-probe control.
7

pancreatitis and healthy controls by intravenous injection. After allowing the
probes to circulate for 5 hours, the pancreata were removed and imaged for Cy5
fluorescence (Fig 6A). With both probes, the Cy5 signal in the inflamed pancreas
was significantly increased compared to normal controls (Fig 6A-B). To
determine whether the fluorescent signal was due to specific binding of
proteases, we analyzed the tissues biochemically (Fig 6C). However, no
protease labeling was observed with either probe. Some autofluorescent
background signal was observed in control samples to which no probe had been
added. Cy5-V-DPP showed an increase in a high molecular weight protein in the
pancreatitis tissues. Its size suggests non-specific binding to a serum protein.
The lack of clear labeling in the 25-30 kDa range suggests that the increase in
fluorescent signal in the whole pancreas was due to non-specific accumulation of
the probes. This may be due to increased blood flow to the inflamed pancreas or
less clearance of unbound probe. While it is obvious that the probes do arrive at
the pancreas, the lack of protease labeling indicates that the probes are not
stable enough to detect in vivo protease activity.
In summary, we have synthesized two fluorescent ABPs for serine proteases and
assessed their utility in vitro and in vivo. The fluorescent Cy5 moiety allows for
rapid, in-gel assessment of protease labeling without the need for membrane
transfer and subsequent detection of biotin with tagged streptavidin conjugates.
Both probes efficiently labeled serine proteases, either purified or in complex
protein mixtures. As anticipated, the activity of trypsin-like proteases was
increased during pancreatitis. Neutrophil elastase activity, on the other hand, was
not strongly detected in the pancreas and colonic luminal fluids, whether healthy
or inflamed. Using exogenously added elastase, we have provided evidence that
elastase-degrading proteases and inhibitors may be upregulated during
inflammation.
The in vivo efficacy of these probes was less desirable, showing non-specific
binding to serum proteins, but no specific protease labeling. Thus, these probes
are suitable for rapid ex vivo monitoring of serine protease activity. They will be
extremely powerful tools, especially in combination with their biotinylated
analogs, which are essential for affinity purification and proteomic identification of
the specific probe targets.
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Labeling recombinant proteases with ABPs
For the ABP dose curve experiment, recombinant proteases were diluted in 20
µL of phosphate-buffered saline: trypsin (100 ng), neutrophil elastase (1 µg),
proteinase-3 (1 µg). The indicated ABP was added from 100x DMSO stocks to
yield final concentrations of 0, 0.1, 0.5, and 1 µM with 1% DMSO. Samples were
incubated for 30 min at 37°C and then solubilized with 4x sample buffer (40%
glycerol, 200 mM Tris-Cl [pH 6.8], 8% SDS, 0.04% bromophenol blue, 5% beta-
mercaptoethanol). Proteins were resolved on a 15% SDS-PAGE gel. To detect
probe-labeled proteins, the gel was scanned for Cy5 fluorescence using a
10

Typhoon flatbed laser scanner (GE Healthcare). For the protease panel, 500 ng
of the indicated protease was labeled and analyzed as above with 1 µM of each
probe: chymotrypsin, neutrophil elastase (Elastin Products Company); trypsin,
plasmin, proteinase-3, thrombin (Sigma); granzyme B (R&D); cathepsin S (gift
from Medivir). Bone marrow was obtained by flushing femurs and tibias of normal
mice with PBS. Cells were sonicated in PBS, and 60 µg of total protein (as
measured by BCA assay) were analyzed as above with the indicated probe at 1
and 10 µM.
Immunoprecipitation
Probe-labeled lysates were divided into input and immunoprecipitation (IP)
samples (100 µg each). The pulldown sample was diluted in 500 µL IP buffer
(PBS [pH 7.4], 1mM EDTA, 0.5% NP-40) along with 5 µL sheep anti-mouse
neutrophil elastase/ELA2 antibody (R&D). Protein A/G beads (40 µl slurry) were
washed with IP buffer and then added to the sample. Tubes were rocked
overnight at 4°C. Beads were washed four times with IP buffer and once with
0.9% sodium chloride. After the last wash, all liquid was removed and proteins
were released from the beads by boiling in 2x sample buffer (20 µl).
Supernatants were then analyzed by fluorescent SDS-PAGE as above.
Acute pancreatitis model
All animal experiments were approved by the Animal Ethics Committee of
Monash University. Eight-week old male C57BL/6J mice were given 12 hourly
injections of caerulein (50 µg/kg in PBS) or PBS12. After the ninth injection, the
indicated ABP or vehicle control was administered intravenously (20 nmol
dissolved in 100 µl 20% DMSO/PBS). Five hours later, pancreata were removed
and imaged for Cy5 fluorescence using an IVIS Spectrum (Perkin Elmer).
Radiance efficiency was determined using Living Image software. Pancreata
were lysed by sonication in PBS, and supernatants were cleared by
centrifugation. Total protein (100 µg) was analyzed by fluorescent SDS-PAGE
analysis as above. Alternatively, lysates from the mice that did not receive an
ABP injection (no probe controls) were analyzed ex vivo as above. Cy5-PK-DPP
and Cy5-V-DPP were used at 10 µM.
Acute colitis model
Eight-week old male C57BL/6J mice were administered 3% dextran sodium
sulfate in the drinking water for 7 days. Colons were excised and flushed with 1
mL cold PBS. Supernatants were cleared by centrifugation. Total protein (50 µg)
was labeled with Cy5-PK-DPP or Cy5-V-DPP and analyzed by fluorescent SDS-
PAGE as above. Alternatively, elastase activity was measured with the
fluorogenic
substrate
probe
MeOSucc-Ala-Ala-Pro-Val-7-amino-4-
methylcoumarin (Bachem, AAPV-AMC). Protein (50 µg) was incubated in the
presence and absence of recombinant neutrophil elastase (1 µg) in a 96-well
format. AAPV-AMC (150 µM final) was added and fluorescence was measured at
10-second intervals over 30 minutes at 360/440 nm excitation/emission. Slopes
were determined and reported.
11

Cy5-V-DPP stability test
Recombinant neutrophil elastase (rNE; 1 or 10 µg as indicated) was aliquoted
alone or spiked into lysates prepared from normal or inflamed pancreata or
luminal fluids (100 µg total protein) followed by immediate addition of Cy5-V-DPP
for 30 min at 37°C or addition after 10 minutes. All samples were then analyzed
using fluorescent SDS-PAGE as above and then analyzed for total elastase
levels by western blot (R&D AF4517; 1:1000) and donkey anti-sheep-IR800
(LiCOR; 1:10,000).
Statistics
All experiments were performed in triplicate using at least 3 biological replicates.
Data are reported as means +/- SEM. Statistical significance was determined by
comparing 2 groups using the Student’s t test, and p values of less than 0.05
were considered significant.
ACKNOWLEDGEMENTS
LEM is supported by an Early Career Fellowship from the National Health and
Medical Research Council (NHMRC, Australia, GNT1091636). BG is the
recipient of an Australian Research Council (ARC) Future Fellowship
(FT130100838). NB is supported by NHMRC and ARC Centre of Excellence in
Convergent Bio-Nano Science and Technology. Work in the authors’ laboratory
is funded in part by Takeda Pharmaceuticals Inc.
12

HO3S
HO3S
N
N+
H2N
Cy5-V-DPP
Cy5-PK-DPP
O
O
OPh
OPh
O
O
P
N
N
H
N
N
OPh
OPh
N
H
P
HO3S
HO3S
O