Strategy for dual-analyte luciferin imaging: In vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation

Article abstract of DOI:10.1021/ja309078t

In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H₂O₂-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-d-Cys (IETDC), which releases d-cysteine in the presence of active caspase 8. Once released, HCBT and d-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H₂O₂ and caspase 8 activity. Using these probes, chemoselective imaging of either H ₂O₂ or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H₂O₂ and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.

Full text of DOI:10.1021/ja309078t

Article
pubs.acs.org/JACS
Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence
Detection of Hydrogen Peroxide and Caspase Activity in a Murine
Model of Acute Inflammation
Genevieve C. Van de Bittner,† Carolyn R. Bertozzi,†,‡,§ and Christopher J. Chang*,†,‡,§
‡
§
†Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California,
Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: In vivo molecular imaging holds promise for
understanding the underlying mechanisms of health, injury,
aging, and disease, as it can detect distinct biochemical
processes such as enzymatic activity, reactive small-molecule
fluxes, or post-translational modifications. Current imaging
techniques often detect only a single biochemical process, but,
within whole organisms, multiple types of biochemical events
contribute to physiological and pathological phenotypes. In
this report, we present a general strategy for dual-analyte
detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can
be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-
2), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive
oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of
active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and
only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the
simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8
activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent
increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a
potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals
during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-
based bioluminescence imaging technologies.
luminescence imaging of hydrogen peroxide (H2O2) fluxes in
living animals, which we utilized to image elevations in the
levels of this reactive oxygen species (ROS) in a murine model
of prostate cancer.25
INTRODUCTION
■
Molecular imaging is an emerging field at the interface of
chemistry and biology that promises to illuminate the
mechanism of a variety of diseases, including cancer,
cardiovascular disorders, diabetes, and Alzheimer’s and related
neurodegenerative diseases.1−7 In this context, firefly luciferin
bioluminescence imaging, in which light is produced by a firefly
luciferase-catalyzed reaction, is an attractive modality, partic-
ularly for in vivo preclinical applications, owing to its red-shifted
emission for good tissue penetration, biological compatibility,
and low background signal.8−11 Indeed, many innovative
molecular probes have been reported that utilize caged luciferin
derivatives to detect a single specific type of biological process,
ranging from enzymatic activity,12−24 to reactive small-molecule
fluxes,25 and post-translational modifications,26 with applica-
tions that span from high-throughput assays to live-cell and live-
animal imaging. In addition, elegantly engineered luciferin
substrates and luciferase enzymes for potential multicolor
applications have been presented27−39 and recently reviewed.40
Our laboratories have contributed to this growing literature by
developing Peroxy Caged Luciferin-1 (PCL-1), a boronate-
caged luciferin that can be used for chemoselective bio-
In addition to changes in ROS, the vast majority of injury,
aging, and disease states have complex alterations in a
combination of biological processes. Thus, we sought to
expand the utility of bioluminescence imaging by using firefly
luciferin to simultaneously monitor multiple biochemical events
in living animals. In this report, we present a general strategy to
visualize two different biochemical processes in vivo via selective
unmasking of a pair of complementary luciferin precursors,
which leads to in situ luciferin formation if and only if both
biochemical processes proceed. This dual-analyte imaging
system relies on a condensation reaction between a
cyanobenzothiazole and cysteine to form luciferin, and in this
context we note related elegant studies that exploit this ligation
for biomolecule labeling purposes.41−44 When applied to dual-
analyte imaging, the cyanobenzothiazole/cysteine condensation
Received: September 12, 2012
Published: January 25, 2013
© 2013 American Chemical Society
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reaction provides an AND-type molecular logic gate, and we
apply it herein to the concurrent and selective detection of
H2O2 and caspase 8 activity, two major contributors to
oxidative stress and cell death in injury and disease.45−54
Peroxy Caged Luciferin-2 (PCL-2) is a boronate-caged probe
that generates 6-hydroxy-2-cyanobenzothiazole (HCBT) upon
reaction with H2O2, and the pentapeptide probe z-Ile-Glu-
ThrAsp-D-Cys (IETDC) produces D-Cys upon caspase 8
cleavage (Figure 1). Application of either probe in conjunction
more recently, for functional detection of enzymatic activity,
small-molecule fluxes, and post-translational modifications, has
been widely adopted owing to its biological compatibility, red-
shifted emission, and high signal-to-noise ratio. Our design
strategy for dual-analyte detection using luciferin biolumines-
cence relies on these aforementioned attractive characteristics,
as well as in situ luciferin formation via a facile cyclization
reaction between HCBT and D-cysteine (Figure 1). This
reaction occurs quickly in aqueous solution and may be
involved in luciferin biosynthesis,55−57 making it ideal for use in
complex biological environments, as demonstrated by use of
this condensation reaction for biomolecule labeling.41−44
Furthermore, in situ luciferin formation affords the opportunity
to create an AND-type molecular logic gate through the
development of a pair of distinct yet complementary probes,
one using a masked HCBT scaffold and one using a masked D-
cysteine precursor, to detect two different types of biological
analytes in an orthogonal fashion. Indeed, in situ luciferin
formation and concomitant production of bioluminescent
signal occurs only if both HCBT as well as the D-cysteine are
released from their respective cages, while unmasking of either
HCBT or D-cysteine alone does not generate appreciable
amounts of free luciferin (Figure 1). To illustrate this concept,
we sought to apply this dual-analyte detection system to the
visualization of specific biological processes, ROS production
and caspase activity, in acute inflammation. Thus, we developed
one probe for detection of H2O2, which is involved in early and
late inflammatory response to external stress and patho-
gens,58−64 and a second probe for caspase 8 detection, owing
to its pivotal role in initiation of the apoptotic pathway during
inflammatory cascades.46,65,66
Figure 1. Design strategy for simultaneous detection of H2O2 and
caspase 8 activity through release of HCBT and D-cysteine and in situ
formation of firefly luciferin.
In designing a luciferin precursor probe for H2O2, we utilized
a general reaction-based method exploited by our lab25,67−78
and others79−86 for chemoselective H2O2 detection, namely the
H2O2-triggered oxidation of boronates to phenols. To this end,
attachment of an aryl boronic acid cage to the phenolic position
of HCBT through a self-immolative linker affords PCL-2 (1,
Scheme 1). For detection of caspase 8, we were inspired by
with the complementary, uncaged luciferin precursor, HCBT or
D-Cys, can be utilized to selectively monitor H2O2 or caspase 8
activity individually, and elevations in both H2O2 and caspase 8
activity lead to increased in situ luciferin formation and
bioluminescent signal when both probes are used in tandem.
We demonstrate that these probes are capable of selectively
monitoring H2O2 and/or caspase 8 activity in aqueous solution
as well as in living cells and animals. Finally, we have applied
this dual-analyte imaging strategy to simultaneously monitor
changes in both H2O2 production and caspase 8 activity in a
murine model of acute inflammation. This work establishes a
logic-gated molecular imaging platform for dual monitoring of
ROS and caspase activity in stages of injury, aging, and disease
that is complementary to multicolor imaging of independent
processes, but avoids the difficulties of spectral unmixing. This
work also provides a starting point for a potentially diverse
array of bioluminescent probes for in vivo multianalyte imaging
through caging of complementary luciferin precursors.
Specifically, other functional groups present on the HCBT
and D-cysteine luciferin precursors could be masked, resulting
in a multianalyte luciferin imaging strategy in which two or
more analytes can be detected simultaneously in the same
animal at the same time, providing an alternative to serial
measurements of single analytes in either the same animal at
different times or two different animals in tandem.
Scheme 1. Synthesis of Peroxy Caged Luciferin-2
previous reports of caspase 8 detection and inhibition, which
rely on the enzymatic cleavage of a short peptide, IETD, to
release a fluorescent molecule or inhibit the enzyme.87−91 For
our purposes, we sought to unmask D-cysteine, which can react
with the HCBT released from PCL-2 to form luciferin in situ.
As such, we prepared the peptide probe IETDC (Figure 1)
containing either free carboxylic acids on the glutamate and
aspartate residues for in vitro studies or esterase-cleavable
methyl ester groups for in vivo studies. In a broader sense, we
RESULTS AND DISCUSSION
■
Design and Synthesis of Peroxy Caged Luciferin-2
and z-Ile-Glu-ThrAsp-D-Cys Probes for Dual-Analyte
Luciferin Imaging. The use of firefly bioluminescence for in
vivo optical imaging of gene expression, tumor growth, and
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Figure 2. Comparison of HCBT/D-cysteine and luciferin. (a) Total bioluminescent signal, integrated over 45 min, from luciferin (0.5−10 μM). (b)
Total bioluminescent signal, integrated over 45 min, from HCBT and D-cysteine (5−25 μM, each) following incubation for 1 h in Tris buffer (50
mM, pH 7.4). To measure luciferin formation in (a) and (b), 100 μg/mL luciferase in 50 mM Tris buffer with 10 mM MgCl2, 0.1 mM ZnCl2, and 2
mM ATP (pH 7.4) was added to the luciferin and HCBT/D-cysteine solutions. (c) Line graph representation of (a), which indicates a linear increase
(R2 = 0.9864) in bioluminescent signal from luciferin (0.5−10 μM). (d) Line graph representation of (b), which indicates an exponential increase
(R2 = 0.9889) in bioluminescent signal from HCBT and D-cysteine (2.5−25 μM each). (e) Total photon flux, integrated over 2 h, from PC3M-luc
cells with HCBT and D-cysteine (0−500 μM, dark gray bars) or luciferin (0−500 μM, light gray bars) in HBSS (25 mM glucose). (f) Representative
image of PC3M-luc cells with HCBT and D-cysteine or luciferin, log scale. (g) Total photon flux, 0−60 min post-injection, for mice injected with
HCBT and D-cysteine (0.05 or 0.5 μM, each) or luciferin (0.05 μM). (h) Representative image (30 min post-injection) of mice injected with HCBT
and D-cysteine or luciferin, log scale. Error bars are SEM; for E, n = 3; for G, n = 3−4.
highlight that the synthesis of both types of luciferin precursor
probe is accessible and versatile, particularly with regard to
peptide-based probes. We envision that masking HCBT or its
amino derivative, as well as D-cysteine and related precursors, at
the amino, sulfhydryl, or carboxylate positions offers ample
opportunities to prepare and mix-and-match a diverse array of
potential probes for multianalyte detection.
Characterization of in Situ Luciferin Formation. To
determine the utility of the in situ luciferin formation approach
for dual-analyte imaging, we compared signals from HCBT/D-
cysteine to luciferin, determined the effect of endogenous L-
cysteine, and analyzed the lifetime of the complementary
HCBT and D-cysteine partner molecules in cells. We first
completed an in vitro test comparing the bioluminescent signal
produced by luciferin to that produced by a mixture of HCBT
and D-cysteine (Figure 2a,b). A linear regression analysis of the
data indicates a linear fit (R2 = 0.9864) for luciferin, while
HCBT/D-cysteine has an exponential fit (R2 = 0.9889), which is
indicative of the second-order reaction between HCBT and D-
cysteine for luciferin formation. Using the luciferin standard
curve (Figure 2a), we determined that 25 μM HCBT/D-
cysteine produces ca. 6.7 μM luciferin, a 27% yield, in vitro.
Subsequent determination of in situ luciferin formation in
PC3M-luc cells indicates that at lower concentrations
exogenous luciferin produces a greater signal than HCBT/D-
cysteine (Figure 2e,f). However, at higher concentrations,
HCBT/D-cysteine produces a greater signal than luciferin. As
HCBT and D-cysteine are not able to produce more luciferin,
and thus bioluminescent signal, than equivalent concentrations
of luciferin, we sought to determine whether the greater
HCBT/D-cysteine signal was due to differences in cell-
membrane permeability. Indeed, when the same comparison
of HCBT/D-cysteine and luciferin is completed following cell
lysis (Figure S1), the signal from HCBT/D-cysteine ap-
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Figure 3. Determination of bioluminescent signal from endogenous L-cysteine. (a) Total photon flux, integrated over 2 h, from PC3M-luc cells with
HCBT (0−50 μM) in the presence (solid line, y1 axis) or absence (dashed line, y2 axis) of D-cysteine (0−50 μM) in HBSS (25 mM glucose). (b)
Representative image of PC3M-luc cells with HCBT D-cysteine, log scale. (c) Total photon flux, 0−60 min post-injection, for mice injected with D-
cysteine (0.05 μmol in 20 μL of PBS) or vehicle (20 μL of PBS) and HCBT (0.05 μM in50 μL of 1:1 DMSO:PBS). (d) Representative image (30
min post-injection) of mice injected with HCBT D-cysteine. Error bars are SEM; for A, n = 3; for C, n = 3−4.
proaches, but does not surpass, the luciferin signal at high
concentrations, indicating that the greater signal from HCBT/
D-cysteine detected within intact, living cells is due to an
increased permeability of HCBT and D-cysteine across the cell
membrane compared to the full luciferin substrate. A final
experiment comparing HCBT/D-cysteine in FVB-luc+ mice,92
which ubiquitously express firefly luciferase, indicates that 0.05
μmol of luciferin produces a ca. 40-fold brighter signal than
0.05 μmol of HCBT/D-cysteine; however, a 10-fold increase in
HCBT/D-cysteine results in a bioluminescent signal that is
equivalent to the 0.05 μmol luciferin signal.
Next, we determined the effect of endogenous L-cysteine on
the signal from HCBT, as previous work documents that firefly
luciferase can convert nonluminescent L-luciferin to lumines-
cent D-luciferin.57 Initial experiments in PC3M-luc cells indicate
a negligible production of bioluminescence from HCBT and
endogenous L-cysteine compared to the signal obtained from
mixing HCBT and D-cysteine (Figure 3a,b). Analogous studies
in FVB-luc+ mice give the same result, as the bioluminescent
signal from the condensation of HCBT and endogenous L-
cysteine is negligible compared to the signal produced when D-
cysteine is present (Figure 3c,d).
Finally, to determine the length of time that released HCBT
and D-cysteine are available for reaction with their comple-
mentary luciferin-forming reagent, cellular lifetime experiments
were completed. Specifically, PC3M-luc cells were first
incubated with HCBT or D-cysteine for 30 min, then the
cells were washed, and then the complementary luciferin-
forming reagent was added after 0, 15, 30, 45, or 60 min. For
the HCBT lifetime studies, we observed that as the time
between removal of HCBT and addition of D-cysteine
increased, there was a decrease in luminescence (Figure
4a,b). This result is likely due to reactions of HCBT with
intracellular nucleophiles41 that make HCBT unavailable for
reaction with D-cysteine. In contrast to HCBT, D-cysteine is still
fully available for reaction with HCBT even an hour after the
cells have been washed (Figure 4c,d), indicating that D-cysteine
has a long lifetime in cells. Notably, this feature could be
exploited for studying healthy and disease states in which one
analyte is produced prior to the second analyte. Taken together,
these analyses illuminate the robust in situ luciferin formation of
HCBT and D-cysteine, the negligible impact of endogenous L-
cysteine, and the cellular availability of HCBT and D-cysteine
for dual-analyte detection.
Peroxide Reactivity and Selectivity of PCL-2. With
these data in hand, we proceeded to determine the ROS
selectivity of PCL-2 for H2O2, the kinetics of the deprotection
reaction, and the concentration dependence of the response of
PCL-2 to H2O2. Reaction of PCL-2 with a panel of biologically
relevant ROS for 5−60 min, followed by incubation with D-
cysteine for in situ luciferin formation and addition of firefly
luciferase, triggers a ca. 50-fold increase in bioluminescence in
the presence of H2O2, with negligible response to other ROS
(Figure 5a). Furthermore, in the presence of catalase, a selective
H2O2-scavenging enzyme, the turn-on bioluminescent signal
produced in the presence of H2O2 is attenuated (Figure 5a),
thus verifying a robust and selective response to H2O2. In
addition, when the control compound HCBT is incubated with
the same panel of ROS, no bioluminescence turn-on is detected
(Figure S2), confirming that the response of PCL-2 to H2O2 is
dependent on reaction at the boronate switch.
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are added to PC3M-luc cells stably transfected with firefly
luciferase. Determination of the resultant bioluminescent signal
with a sensitive charge-coupled device (CCD) camera again
indicates a linear response (R2 = 0.9993) to H2O2 over 2 orders
of magnitude, from 0 to 100 μM H2O2 (Figure 5d−f). Taken
together, the selective response of PCL-2 to H2O2, as well as its
dose-dependent turn-on, low micromolar detection limit, and
ability to form luciferin in situ show that PCL-2 possesses the
requisite features for in vivo detection of alterations in H2O2
levels in living organisms.
Selective Cleavage of IETDC by Caspase 8. After
determining the selectivity and sensitivity of PCL-2 for turn-on
bioluminescent H2O2 detection, we evaluated the ability of
caspase 8 to cleave IETDC and release D-cysteine for in situ
luciferin formation. This was accomplished via incubation of
IETDC with caspase 8 for 60 min in the presence of HCBT.
After subsequent addition of firefly luciferase, the bio-
luminescent signal was determined, indicating a ca. 27-fold
turn-on response (Figure 6). More importantly, addition of a
broad-spectrum caspase inhibitor, Q-VD-OPh,93,94 shows
complete attenuation of the response of IETDC to caspase 8,
and incubation with other caspase enzyme isoforms that are
important for the inflammatory caspase cascade, caspase 3 and
caspase 9,46 do not give rise to a turn-on bioluminescent
response for the peptide probe (Figure 6). These latter control
experiments verify the chemoselectivity of the IETDC probe
for caspase 8 detection. Finally, incubation of D-cysteine with
these caspase enzymes causes no change in bioluminescent
signal (Figure S4), further indicating that the response seen
with the IETDC luciferin precursor probe is caspase 8-
dependent.
Dual Detection of H2O2 and Caspase 8 Activity via in
Situ Luciferin Formation. To establish the utility of our
probes for dual imaging of H2O2 and caspase 8 activity, we
incubated PCL-2 with H2O2, followed by addition of caspase 8
and IETDC. The resultant bioluminescent signal indicates that
in the presence of these two analytes, both probes are
deprotected, and luciferin is readily formed to produce a ca.
18-fold increase in bioluminescent signal (Figure 7). Notably,
addition of a single analyte, either H2O2 or caspase 8, results in
little to no increase in bioluminescent signal, verifying that the
system acts as an AND-type molecular logic gate by requiring
the presence of both analytes for bioluminescent signal
production. Moreover, treatment with either a H2O2 scavenger,
catalase, or a caspase inhibitor, Q-VD-OPh, attenuates the
increase in signal observed in the presence of H2O2 and
caspase 8, further demonstrating that both probes must be
uncaged for luciferin formation and bioluminescent signal
production (Figure 7). As expected, treatment with both
inhibitors causes a complete attenuation of signal, providing
additional validation for the use of PCL-2 and IETDC for dual-
analyte detection. Finally, several additional sets of control
experiments were performed before moving on to in vivo
imaging experiments. First, in contrast to the turn-on response
of the dual-probe system in the presence of both H2O2 and
caspase 8, incubation of HCBT and D-cysteine with H2O2 and
caspase 8 causes no alteration of the bioluminescent signal
(Figure S5). Also, addition of catalase and Q-VD-OPh does not
interfere with in situ luciferin formation or the resultant
bioluminescent signal (Figure S5). Taken together, the tandem
use of PCL-2 and IETDC for dual-analyte imaging in vitro
highlights this unique method for detection of two different
analytes through production of a single bioluminescent signal.
Figure 4. Lifetime of HCBT and D-cysteine in PC3M-luc cells. (a)
Total photon flux, integrated over 2 h, from PC3M-luc cells with D-
cysteine (100 μM) added 0, 15, 30, 45, or 60 min after incubation with
HCBT (100 μM). (b) Representative image of PC3M-luc cells with D-
cysteine added 0, 15, 30, 45, or 60 min following removal of HCBT.
(c) Total photon flux, integrated over 2 h, from PC3M-luc cells with
HCBT (100 μM) added 0, 15, 30, 45, or 60 min after incubation with
D-cysteine (100 μM). (d) Representative image of PC3M-luc cells with
HCBT added 0, 15, 30, 45, or 60 min following removal of D-cysteine.
Error bars are SEM; for A and C, n = 3.
Next, we analyzed the kinetics and dose dependence of the
PCL-2 and H2O2 reaction. A comparison of the measured
second-order rate constant (k = 2.7 M−1 s−1, Figure S3) for the
PCL-2 and H2O2 reaction to the catalytic constant (kcat = 1.6
s−1) for firefly luciferase29 indicates that release of HCBT from
PCL-2 is rate-limiting at the micromolar concentrations used in
our studies. The dose dependence of the response of PCL-2 to
H2O2 was then determined via incubation of PCL-2 with
various concentrations of H2O2 (0−100 μM) for 60 min, prior
to incubation with D-cysteine and addition of firefly luciferase.
As shown in Figure 5b,c, PCL-2 shows a linear response (R2 =
0.9957) to H2O2 over 2 orders of magnitude, from 1 to 100 μM
H2O2. Furthermore, this same response is found in cellulo when
PCL-2 (25 μM), D-cysteine (25 μM), and H2O2 (0−100 μM)
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Figure 5. Selective and concentration-dependent bioluminescent detection of H2O2 by PCL-2. (a) Total bioluminescent signal, integrated over 10
min, from PCL-2 (5 μM) alone (light gray bars) or incubated with various ROS (100 μM) or H2O2 (100 μM) and catalase (0.4 mg/mL) for 5, 20,
40, or 60 min. Signals normalized to signal from PCL-2 in the absence of any ROS. (b) Total bioluminescent signal, integrated over 15 min, from 5
μM PCL-2 incubated for 1 h with increasing concentrations of H2O2 (0−100 μM). To measure HCBT release in (a) and (b), PCL-2/ROS solutions
were incubated with D-cysteine (20 μM) for 15 min, prior to addition of 100 μg/mL luciferase in 50 mM Tris buffer with 10 mM MgCl2, 0.1 mM
ZnCl2, and 2 mM ATP (pH 7.4). (c) Line graph representation of (b), which indicates a linear increase (R2 = 0. 9957) in bioluminescent signal from
PCL-2 in the presence of H2O2 in aqueous solution. (d) Total photon flux, integrated over 2 h, from PC3M-luc cells with PCL-2 (25 μM), D-
cysteine (25 μM), and H2O2 (0−100 μM) in DMEM. (e) Line graph representation of (d), which indicates a linear increase (R2 = 0.9993) in
bioluminescent signal from PCL-2 in the presence of H2O2 in PC3M-luc cells. (f) Representative image of PC3M-luc cells with PCL-2, D-cysteine,
and H2O2 in DMEM, log scale. Statistical analyses were performed with a two-tailed Student’s t-test. *P < 0.01 (for D and E, n = 6), and error bars
are SEM.
Figure 6. Selective and sensitive bioluminescent detection of caspase 8
activity by IETDC. Total bioluminescent signal, integrated over 10
min, from IETDC (5 μM) and HCBT (5 μM) alone or incubated with
various caspase enzymes (3 and 8: 1 unit; 9: 0.001 unit) or caspase 8
and Q-VD-OPh (10 μM) for 60 min. From left to right, 1: IETDC and
HCBT; 2: IETDC, HCBT, and caspase 8; 3: IETDC, HCBT,
Figure 7. Dual detection of H2O2 and caspase 8 via in situ luciferin
formation. Total bioluminescent signal, integrated over 10 min, from
PCL-2 (10 μM) and IETDC (10 μM) alone or incubated with H2O2
(250 μM) and caspase 8 (1 unit) in the presence or absence of catalase
(1 unit) and/or Q-VD-OPh (10 μM). From left to right, 1: PCL-2 and
IETDC; 2: PCL-2, IETDC, and H2O2; 3: PCL-2, IETDC, and
caspase 8, and Q-VD-OPh; 4: IETDC, HCBT, and caspase 3; 5:
caspase 8; 4: PCL-2, IETDC, H2O2, and caspase 8; 5: PCL-2, IETDC,
IETDC, HCBT, and caspase 9. Signals normalized to signal from
H2O2, caspase 8, and catalase; 6: PCL-2, IETDC, H2O2, caspase 8, and
IETDC and HCBT in the absence of the caspase enzymes. To
Q-VD-OPh; 7: PCL-2, IETDC, H2O2, caspase 8, catalase, and Q-VD-
OPh. Signals normalized to signal from PCL-2 and IETDC in the
absence of H2O2 and caspase 8. To quantify luciferin formation, 100
μg/mL luciferase in 50 mM Tris buffer with 10 mM MgCl2, 0.1 mM
quantify luciferin formation, 100 μg/mL luciferase in 50 mM Tris
buffer with 10 mM MgCl2, 0.1 mM ZnCl2, and 2 mM ATP (pH 7.4)
was added to 100 μL of each IETDC/HCBT solution.
ZnCl2, and 2 mM ATP (pH 7.4) was added to the PCL-2/IETDC
solutions.
Molecular Imaging of H2O2 Fluxes in Living FVB-luc+
Mice with PCL-2. Following the in vitro demonstration of the
utility of PCL-2 and IETDC for dual-analyte imaging, we next
sought to apply PCL-2 to molecular imaging of H2O2 fluxes in
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Figure 8. Bioluminescent response of PCL-2 to H2O2 in FVB-luc+ mice. (a) Representative image (10 min post-injection) for mice injected with a
mixture of PCL-2 and D-cysteine (i.p., 0.05 μmol each, in 50 μL of 1:1 DMSO:PBS) immediately prior to injection of H2O2 (i.p., 0, 0.5, 1.5, or 4.5
μmol, left to right, in 100 μL of PBS). (b) Total photon flux, 0−15 min post-injection, for mice injected with PCL-2 and D-cysteine H2O2. (c)
Representative image (10 min post-injection) for mice injected with NAC (i.p., 10 mg/kg in 25 μL of PBS, pH 7−8) or PBS (i.p., 25 μL) two min
prior to injection of a mixture of PCL-2 and D-cysteine (i.p., 0.05 μmol each, in 50 μL of 1:1 DMSO:PBS) and a solution of H2O2 (i.p., 1.5 μmol in
75 μL of PBS). (d) Total photon flux, 0−15 min post-injection, for mice injected with PCL-2 and H2O2 in the presence or absence of NAC.
Statistical analyses were performed with a two-tailed Student’s t-test. *P < 0.05, **P < 0.01 (for B, n = 4; for D, n = 3), and error bars are SEM.
FVB-luc+ mice that ubiquitously express firefly luciferase.92 For
our initial in vivo studies, several doses of H2O2 were injected
into the intraperitoneal (i.p.) cavity of mice, along with a
solution of PCL-2 and D-cysteine. The animals were then
imaged using a CCD camera to detect the luciferin formed
following deprotection of PCL-2 by H2O2 and in situ cyclization
with D-cysteine. These imaging experiments reveal a detection
limit of ca. 0.5 μmol of H2O2 and a robust increase in luciferin
production as a function of H2O2 dose, with a ca. 10-fold turn-
on in bioluminescent signal following treatment with 4.5 μmol
of H2O2 (Figure 8). Comparison of PCL-2 to PCL-1 imaging
under optimized imaging conditions for each probe indicates
that PCL-1 has a lower detection limit for H2O2 than PCL-2 in
vivo (0.037 μmol versus 0.5 μmol), while PCL-2 shows an
increased fold turn-on when higher amounts of H2O2 are
injected (6.4-fold with 1.5 μmol of H2O2 for PCL-2 versus 3.6-
fold with 2.4 μmol of H2O2 for PCL-1; Table S1).
In additional experiments to further show that H2O2 is
required for the turn-on bioluminescent response, a separate
group of mice was treated with 1.5 μmol of H2O2 in the
presence or absence of N-acetyl-L-cysteine (NAC), a small-
molecule H2O2 scavenger.95 In the absence of NAC, a robust
bioluminescent signal is detected; however, upon NAC
addition the bioluminescent signal is considerably attenuated
(Figure 8), thus providing evidence that the signal increase
observed with H2O2 injection is a result of reaction of the PCL-
2 probe with H2O2 in vivo. Finally, additional control
experiments with a solution of HCBT and D-cysteine showed
no change in the luciferin bioluminescent signal upon
treatment with either H2O2 or NAC (Figure S6).
on the results from these in vitro and in vivo experiments, we
next applied the PCL-2 and IETDC probes to the individual
detection of their respective bioanalytes in a murine model of
acute inflammation. As stated previously, both H2O2 and
caspase 8 play important roles in the development and
progression of the inflammatory response, wherein H2O2 has
been found to be important for eradication of pathogens,59,60,96
as well as playing a role in cellular signaling.58,61−63 Caspase 8 is
important in its own right as one of the key initiators of the
apoptotic cell-death pathway that is often initiated during
inflammation.46,65,66 To monitor these two analytes individu-
ally, an acute inflammatory response was induced via injection
of mice with lipopolysaccharides (LPS), a lipoglycan found on
the surface of pathogenic bacteria that causes an inflammatory
response in vivo.97−100
For H2O2 detection during inflammation, mice were treated
with LPS or vehicle 6 h prior to injection of PCL-2 and D-
cysteine. These studies indicate a ca. 3.7-fold turn-on in
response to LPS stimulation, with a 55% reduction in signal
upon treatment with apocynin101−103 (Figure 9a,b), an
antioxidant and broad-spectrum inhibitor of NADPH oxidase
enzymes that are a major source of ROS production during
inflammation and the general immune response.58,59,63,104 The
increase in bioluminescent signal detected upon LPS treatment,
as well as its attenuation by apocynin, indicate that PCL-2
successfully monitors alterations in endogenous H2O2 levels in
living animals during inflammation. These data are further
supported by previous, imaging-independent studies using ex
vivo analysis of tissues or cells, which have shown an increase in
ROS following LPS injection under similar conditions as
demonstrated by lipid peroxidation and an increased oxidized
to reduced glutathione ratio.105−107
In Vivo Detection of Endogenous H2O2 Fluxes and
Caspase 8 Activity during Acute Inflammation. Building
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Figure 9. Bioluminescent signal from PCL-2 and IETDC following lipopolysaccharide challenge. (a) Representative image (10 min post-injection)
of mice 6 h after injection of LPS (i.p., 3 mg/kg in 50 μL of saline) or saline vehicle (i.p., 50 μL). Two minutes prior to imaging, mice were treated
with apocynin (i.p., 10 mg/kg in 20 μL of DMSO) or vehicle (i.p., 20 μL of DMSO). Imaging was started following injection of a mixture of PCL-2
and D-cysteine (i.p., 0.05 μmol each, in 50 μL of 1:1 DMSO:PBS). (b) Total photon flux, 0−15 min post-injection, for mice injected with PCL-2 and
D-cysteine, LPS, and apocynin. (c) Representative image (30 min post-injection) of mice 6 h after injection of LPS (i.p., 3 mg/kg in 50 μL of
saline) or vehicle (i.p., 50 μL of saline). Thirty minutes prior to imaging, mice were treated with z-VD(OMe)-OPh (i.p., 1 μmol in 20 μL of DMSO)
or vehicle (i.p., 20 μL of DMSO). Imaging was started following injection of a mixture of IETDC and HCBT (i.p., 0.05 μmol each, in 50 μL of 1:1
DMSO:PBS). (d) Total photon flux, 15−45 min post-injection, for mice injected with IETDC and HCBT, LPS, and z-VD(OMe)-OPh.
Statistical analyses were performed with a two-tailed Student’s t-test. *P < 0.05 (for B and D, n = 3−4), and error bars are SEM.
Figure 10. Dual imaging of H2O2 and caspase 8 following lipopolysaccharide challenge. (a) Representative image (45 min post-injection) of mice
following injection of ascorbic acid (i.p., 200 mg/kg in 30 μL of saline), LPS (i.p., 3 mg/kg in 50 μL of saline), z-VD(OMe)-OPh (i.p., 1 μmol in 20
μL of DMSO), and/or their respective vehicles (i.p., 30 or 50 μL of saline or 20 μL of DMSO). Mice were treated with ascorbic acid or vehicle 4.5 h
prior to imaging, LPS or vehicle 4 h prior to imaging, and z-VD(OMe)-OPh or vehicle 2 h prior to imaging. Imaging was started following injection
of a mixture of PCL-2 and IETDC (i.p., 0.05 μmol each, in 50 μL of 7:3 DMSO:PBS). (b) Total photon flux, 30−60 min post-injection, for mice
injected with PCL-2 and IETDC, LPS, z-VD(OMe)-OPh, and ascorbic acid. Statistical analyses were performed with a two-tailed Student’s t-
test. *P < 0.05 (for B and D, n = 3−4), and error bars are SEM.
Parallel studies were completed with IETDC to verify its
ability to monitor caspase 8 activity during acute inflammation
in this same murine model. Treatment of FVB-luc+ mice with
LPS or vehicle 6 h prior to injection of IETDC and HCBT
reveals an even greater increase in bioluminescent signal of ca.
18-fold (Figure 9c,d). Furthermore, a 34% attenuation of the
signal is afforded by injection of the pan-caspase inhibitor z-
VD-OPh, a methyl ester protected analog of Q-VD-OPh, thus
establishing that IETDC reliably monitors increased caspase 8
activity following LPS stimulation (Figure 9c,d). Again, this
increase in caspase 8 activity upon LPS stimulation is supported
by ex vivo tissue studies that indicate increased caspase 8
expression and cleavage of procaspase 8 to form active caspase 8
following treatment with LPS.91,108,109
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To verify that both apocynin and z-VD-OPh have no affect
on the luciferin bioluminescent signal, a solution of HCBT and
D-cysteine was injected following apocynin, z-VD-OPh, or
vehicle injection. Detection of the resultant bioluminescent
signal indicates that apocynin and z-VD-OPh do not alter the
luciferin signal in vivo (Figure S7). Taken together, these data
indicate an increase in both the level of H2O2 and the activity of
caspase 8 in living mice during acute inflammation.
In Vivo Dual-Analyte Detection of Endogenous H2O2
and Caspase 8 Activity during Acute Inflammation. After
confirming the individual abilities of the PCL-2 and IETDC
probes to detect alterations in endogenous H2O2 levels and
caspase 8 activity, respectively, in living animals in an
inflammation disease model, we next applied these two probes
for simultaneous detection of both bioanalytes in vivo. For these
studies, LPS was again used to provoke an acute inflammatory
response in FVB-luc+ mice. Initial experiments with PCL-2 and
IETDC in control animals not treated with LPS clearly
demonstrate that injection of both probes results in a very low
bioluminescent signal (Figure 10), indicating very low basal
levels of H2O2 and/or caspase 8 activity. To our delight,
induction of inflammation via treatment with LPS causes a 2.7-
fold increase in bioluminescent signal (Figure 10), verifying use
of this luciferin-based molecular logic gate system to indicate
the simultaneous presence of both H2O2 and caspase 8 during
acute inflammation.
We then moved on to examine whether the turn-on signal for
the dual-analyte molecular logic gate system could be
attenuated by reducing ROS and caspase 8 activity. For these
experiments, pretreatment with the antioxidant ascorbic acid
(vitamin C) was used to reduce the level of H2O2, as previous
literature suggests a potential clinical application of this
antioxidant for treatment of sepsis and other inflammatory
responses.110−112 The caspase inhibitor z-VD-OPh was used for
reduction of caspase 8 activity. As expected, use of both these
compounds results in a 30% decrease in bioluminescent signal
(Figure 10), indicating the utility of PCL-2 and IETDC for
dual-analyte detection of H2O2 and caspase 8 during acute
inflammation. Importantly, injection of ascorbic acid does not
impact the bioluminescent signal from HCBT and D-cysteine
(Figure S8), demonstrating that this vitamin alone does not
alter the bioluminescent signal of luciferin. The successful
application of PCL-2 and IETDC in this in vivo model of
inflammation not only demonstrates the ability of both probes
to detect their respective single analytes in living animals but
further establishes the tandem use of PCL-2 and IETDC to
detect the concomitant increase in two different biochemical
events using a single bioluminescent reporter system.
a key small molecule (H2O2) and enzyme activity (caspase 8)
involved in a wide variety of injury and disease states.
Specifically, the H2O2 detection probe PCL-2 relies on
conversion of an aryl boronic acid to a phenol to release one-
half of firefly luciferin, HCBT. Complementary design and
synthesis of a caspase 8 activity probe is based on a cleavable
peptide, IETDC, which reacts with caspase 8 to release D-
cysteine, the other half of firefly luciferin. These probes work
together to form an AND-type molecular logic gate in which
concomitant cleavage of both probes is required to produce a
bioluminescent signal, which results from release of both
HCBT and D-cysteine and subsequent in situ luciferin
formation. We establish that PCL-2 and IETDC can be used
separately to selectively detect their targeted analytes, H2O2 and
caspase 8, respectively, and that they can be used concurrently
in vitro for dual imaging of H2O2 fluxes and caspase 8 activity.
Moreover, subsequent individual use of PCL-2 and IETDC in
vivo during an LPS-induced acute inflammatory response
verifies the ability of these probes to detect alterations in
endogenous levels of H2O2 and caspase 8 activity. Finally, PCL-
2 and IETDC can be used in tandem to simultaneously image
H2O2 fluxes and caspase 8 activity, respectively, in a murine
model of acute inflammation, showing that this molecular logic
gate approach is applicable all the way from in vitro plate-reader
assays to in vivo animal disease models. Importantly, the
background signal following injection of both probes is
extremely low, highlighting the bioorthogonal nature of the
probes and their reliable use in vivo. On balance, we note a
potential limitation of the system as described here is that both
the cyanobenzothiazole and D-cysteine partners must be
unmasked and bioavailable in the same time and space for
luciferin formation within the time frame of imaging, so
quantitation may be challenging owing to spatial and temporal
variations in pharmacokinetics and tissue distributions of caged
species, particularly on the cyanobenzothiazole end. However,
the relatively long lifetime of D-cysteine suggests that caged
versions of this partner may allow imaging of analytes produced
at different time scales. To expand the scope of this
multianalyte detection technology, current efforts are focused
on the development of alternative probes for other biochemical
processes, including enzymatic activities and related stoichio-
metric and catalytic reactions, small molecules and ions, and
post-translational and other covalent and ionic modifications.
EXPERIMENTAL SECTION
■
Synthetic Materials and Methods. Compound 3 was synthe-
sized according to literature procedures.25 Chemicals were purchased
from Sigma-Aldrich (St. Louis, MO), EMD Chemicals Inc. (Gibbs-
town, NJ), Alfa Aesar (Ward Hill, MA), and Thermo Fisher Scientific
(Waltham, MA) and were used as received. Column chromatography
was performed using SiliaFlash P60 silica gel (40−63 μm) from
Silicycle (Quebec, Canada). Analytical thin layer chromatography was
performed using glass-backed SiO2 TLC plates from Silicycle. NMR
spectra were obtained in deuterated solvents from Cambridge Isotope
Laboratories (Cambridge, MA) on a Bruker AV-400 spectrometer at
the College of Chemistry NMR Facility at the University of California,
Berkeley. All chemical shifts are reported in the standard δ notation of
parts per million using the peaks of residual proton and carbon signals
of the solvent as internal references. Low-resolution electrospray
ionization (ESI) mass spectral analyses were performed on an Agilent
6100 series single quad LC/MS system or an Agilent 7890A GC
system with a 5975C inert MSD with a triple-axis detector.
CONCLUDING REMARKS
■
The field of molecular imaging continues to produce an
expanding set of unique chemical tools for studying living
biological systems, thus affording new opportunities to
elucidate the complex processes involved in various states of
health, injury, aging, and disease. Central to this effort is the
development of new detection methods that can be directly
translated from in vitro assays to cellular and in vivo imaging
applications. In this study, we have presented a general method
for monitoring two different biological analytes with a single
bioluminescent reporter system via in situ luciferin formation.
By caging the two complementary luciferin precursors, HCBT
and D-cysteine, with appropriate reactive protecting groups, we
have devised a new sensing system for concomitant detection of
4-((2-Cyanobenzo[d]thiazol-6-yloxy)methyl)phenylboronic
Acid (1). HCBT (3) (150 mg, 0.85 mmol, 1.1 equiv) and 4-
(hydroxymethyl)benzeneboronic acid (4) (166 mg, 0.77 mmol, 1.0
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equiv) were dissolved in 15 mL of dry N,N-dimethylformamide prior
to the addition of cesium carbonate (277 mg, 0.85 mmol, 1.0 equiv).
The mixture was stirred at 60 °C for 45−50 min before it was allowed
to cool to room temperature. Ethyl acetate (EtOAc, 100 mL) was
added to the reaction, and the organic phase was washed with
deionized H2O (3 × 50 mL). The aqueous layers were combined and
back-extracted with EtOAc (3 × 30 mL). All of the organic layers were
combined, washed twice with brine, dried over sodium sulfate, and
concentrated in vacuo. The crude material was purified on a silica
column (90:10 EtOAc:methanol, dry loaded) to give 225 mg (94%) of
the pure product as a white powder. 1H NMR (400 MHz, d6-acetone):
δ 5.33 (2H, s), 7.23 (2H, s), 7.45 (1H, dd, J = 9.0, 2.6 Hz), 7.52 (2H,
d, J = 8.0 Hz), 7.91−7.94 (3H, m), 8.16 (1H, d, J = 8.8 Hz). LRMS
(m/z): [M]+ calcd for C15H12BN2O3S, 311.1; found, 311.1.
Kinetic Analysis. H2O2 (1, 2.5, or 5 mM) was added to PCL-2 (10
μM) in Tris buffer (pH 8.5) and absorbance measurements were
recorded every 15−30 s over 5−20 min to determine the second-order
rate constant for the reaction. Measurements were taken at pH 8.5 to
distinguish the HCBT peak, which shifts from 321 to 379 nm when it
is deprotonated, from the PCL-2 peak (321 nm).
Bioluminescent Assays. Millipore water was used to prepare all
aqueous solutions. Luciferase (100 μg/mL) in 50 mM Tris buffer, pH
7.4, with 10 mM MgCl2, 0.1 mM ZnCl2, and 2 mM ATP was added to
selectivity assay samples (described below) to determine the relative
amount of luciferin formed during incubation. Measurements for
bioluminescent assays were performed at 37 °C and were recorded
using a Molecular Devices SpectraMax M2 plate reader (Sunnyvale,
CA). Samples for bioluminescent measurements were placed in white,
opaque 96-well plates, which were purchased from Corning Inc.
(Corning, NY). ATP was purchased from MP Biomedicals (Solon,
OH) or Sigma-Aldrich (St. Louis, MO), and luciferase was purchased
from Promega (Madison, WI). Caspase enzymes were purchased from
Sigma-Aldrich, and caspase inhibitors were purchased from MP
Biomedicals.
Standard Curve for Luciferin Bioluminescence. Luciferin
(0.5−10 μM) was incubated in 100 μL of Tris buffer (50 mM, pH
7.4) for 60 min prior to addition of 100 μL of a Tris buffer (50 mM,
pH 7.4) containing luciferase (100 μg/mL), 10 mM MgCl2, 0.1 mM
ZnCl2, and 2 mM ATP. Following addition of the luciferase-containing
solution the bioluminescent signal was detected.
100 μL of a Tris buffer (50 mM, pH 7.4) containing luciferase (100
μg/mL), 10 mM MgCl2, 0.1 mM ZnCl2, and 2 mM ATP was added to
100 μL of the PCL-2 solutions and the bioluminescent signal was
detected.
Selectivity Assays for IETDC (Carboxylic Acid). For determi-
nation of caspase selectivity, various caspases (3 and 8, 1 unit; 9, 0.001
unit) were administered to IETDC or D-cysteine (5 μM) in Tris buffer
(50 mM, pH 7.4). A caspase buffer (10% sucrose containing 20 mM
Tris-HCl, 150 mM imidazole-HCl, 20 mM 2-mercaptoethanol, 500
mM NaCl, 2.5 mM EDTA, and 0.1% CHAPS, pH 8.0) was also added
with the caspase enzymes to a total volume (enzyme + buffer) of 5 μL.
Additionally, dithiothreitol (1 mM) and HCBT (5 μM) were added to
each solution for luciferin formation. For experiments with caspase
inhibition, a pan-caspase inhibitor, Q-VD-OPh (10 μM), was added to
the Tris buffer and incubated with caspase 8 for 15 min prior to
delivery of IETDC, DTT, and HCBT. After each enzyme was
incubated with IETDC or D-cysteine for 60 min, 100 μL of a Tris
buffer (50 mM, pH 7.4) containing luciferase (100 μg/mL), 10 mM
MgCl2, 0.1 mM ZnCl2, and 2 mM ATP was added to 100 μL of the
IETDC or D-cysteine solutions and the bioluminescent signal was
detected.
Selectivity Assays for Dual H2O2 and Caspase 8 Detection.
To demonstrate dual imaging of H2O2 and caspase 8 in vitro, H2O2
(250 μM) was first added to a solution of PCL-2 (10 μM) or HCBT
(5 μM) in Tris buffer (50 mM). To quench H2O2 immediately,
catalase (1 unit) was subsequently added to some of the solutions.
After 60 min, catalase (1 unit) was added to all other solutions
containing H2O2 to quench any remaining H2O2. Subsequently,
caspase 8 (1 unit) was added to the solutions in the presence or
absence of the caspase inhibitor, Q-VD-OPh (10 μM), 15 min prior to
IETDC (10 μM) or D-cysteine (5 μM) addition. Following IETDC or
D-cysteine addition, the solutions were incubated for 60 min.
Additionally, dithiothreitol (1 mM) was added to all solutions at the
time of caspase 8 addition to maintain the reduced state of the D-
cysteine. To monitor luciferin formation, 100 μL of a Tris buffer (50
mM, pH 7.4) containing luciferase (100 μg/mL), 10 mM MgCl2, 0.1
mM ZnCl2, and 2 mM ATP was added to 100 μL of the PCL-2/
IETDC or HCBT/D-cysteine solutions and the bioluminescent signal
was detected.
Cellular Assays. A Xenogen IVIS Spectrum instrument (Caliper
Life Sciences, Hopkinton, MA) was used for bioluminescent imaging
in all cellular experiments. PC3M-luc cells (kindly provided by Chris
Contag, Stanford University) were cultured in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS).
Prior to assaying, cells were passed and plated (1.3−1.5 × 104 cells/
well) in black 96-well plates with clear bottoms (Becton Dickinson and
Co., Franklin Lakes, NJ). Once the cells were ca. 95% confluent,
reagents were added for bioluminescent assays.
Comparison of in Situ Luciferin Formation and Luciferin in
PC3M-luc Cells. PC3M-luc cells prepared as described above were
washed with HBSS (25 mM glucose) prior to addition of HCBT and
D-cysteine (0−500 μM, 1% DMSO) or luciferin (0−500 μM, 1%
DMSO) in HBSS (25 mM glucose). For experiments with lysed cells,
100 μL of Glo lysis buffer (Promega, Madison, WI) was incubated
with the PC3M-luc cells for 5 min prior to addition of HCBT and D-
cysteine or luciferin. The plates were immediately imaged for 2 h to
determine the peak bioluminescent signal produced.
In Situ Luciferin Formation from HCBT and D-Cysteine.
HCBT (2.5−25 μM) was incubated with D-cysteine (2.5−25 μM) in
100 μL of Tris buffer (50 mM, pH 7.4) for 60 min prior to addition of
100 μL of a Tris buffer (50 mM, pH 7.4) containing luciferase (100
μg/mL), 10 mM MgCl2, 0.1 mM ZnCl2, and 2 mM ATP. Following
addition of the luciferase-containing solution the bioluminescent signal
was detected.
Selectivity Assays for PCL-2. Various ROS (100 μM) were
administered to PCL-2 or HCBT (5 μM) in Tris buffer (50 mM, pH
7.4) with or without 10 mM MgCl2 and 0.1 mM ZnCl2. Hydrogen
peroxide (H2O2), tert-butyl hydroperoxide (TBHP), and hypochlorite
(−OCl) were delivered from 10 mM stock solutions prepared using
30%, 70%, and 6.15% aqueous solutions, respectively. Hydroxyl radical
(•OH) and tert-butoxy radical (•OtBu) were generated by reaction of
1 mM FeCl2 with 100 μM H2O2 or TBHP, respectively. Nitric oxide
−
(NO•) was delivered using PROLI NONOate, and superoxide (O2 )
was delivered from a 10 mM stock solution of KO2 in DMSO.
Experiments with H2O2 and catalase were performed with 100 μM
H2O2 and 0.4 mg/mL catalase. After each ROS was incubated with
PCL-2 for 5, 20, 40, or 60 min or HCBT for 60 min, dithiothreitol (1
mM) and D-cysteine (20 μM) were added. For solutions with Fe2+,
EDTA (1 mM) was added prior to D-cysteine to chelate the iron. After
an additional 15 min incubation, 100 μL of a Tris buffer (50 mM, pH
7.4) containing luciferase (100 μg/mL), 10 mM MgCl2, 0.1 mM
ZnCl2, and 2 mM ATP was added to 100 μL of the PCL-2 or HCBT
solutions and the bioluminescent signal was detected.
Determination of Signal Produced from Endogenous L-
Cysteine in PC3M-luc Cells. PC3M-luc cells prepared as described
above were washed with HBSS (25 mM glucose) prior to addition of
HCBT and D-cysteine (0−50 μM, 1% DMSO) or HCBT alone (0−50
μM, 1% DMSO) in HBSS (25 mM glucose). The plate was
immediately imaged for 2 h to determine the peak bioluminescent
signal produced.
Lifetime of HCBT and D-Cysteine in PC3M-luc Cells. The
medium was removed from PC3M-luc cells prepared as described
above, and HCBT (100 μM, 1% DMSO) or D-cysteine (100 μM, 1%
PBS) in DMEM (-FBS) was added. After a 30 min incubation, the
HCBT or D-cysteine was removed, the cells were washed with DMEM
(-FBS), and fresh DMEM was added. After 0, 15, 30, 45, or 60 min,
Concentration Dependence of PCL-2. PCL-2 was incubated in
Tris buffer (50 mM, pH 7.4) with various concentrations of H2O2 for
60 min prior to the addition of catalase (0.4 mg/mL), dithiothreitol (1
mM) and D-cysteine (20 μM). After an additional 15 min incubation,
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the second component of luciferin, D-cysteine (100 μM final
concentration, 1% PBS) or HCBT (100 μM final concentration, 1%
DMSO) in HBSS (25 mM glucose) was added, and the plate was
immediately imaged for 2 h to determine the peak bioluminescent
signal produced.
with a mixture of IETDC and HCBT (0.05 μmol each, in 50 μL of 1:1
DMSO:PBS) and imaged.
For studies with PCL-2 and IETDC, mice were injected i.p. with
ascorbic acid (200 mg/kg in 30 μL of saline) or the saline vehicle (30
μL) 30 min prior to i.p. injection of LPS (3 mg/kg in 50 μL of saline)
or saline (50 μL). Two hours after injection of LPS, z-VD(OMe)-OPh
(1 μmol in 20 μL of DMSO) or the vehicle, DMSO (20 μL), was
injected i.p. Two hours later, mice were injected i.p. with a mixture of
PCL-2 and IETDC (0.05 μmol each, in 50 μL of 7:3 DMSO:PBS) and
imaged.
Concentration Dependence of PCL-2 with PC3M-luc Cells.
The medium was removed from PC3M-luc cells prepared as described
above, and PCL-2 (25 μM, 2.5% final DMSO concentration), D-
cysteine (25 μM), and H2O2 (0−100 μM final concentrations) in
DMEM (-FBS) were added. The plate was immediately imaged for 2 h
to determine the peak bioluminescent signal produced from PCL-2.
Animal Experiments. A Xenogen IVIS Spectrum instrument
(Caliper Life Sciences, Hopkinton, MA) was used for bioluminescent
imaging in all animal experiments. Mice were anesthetized prior to
injection and during imaging via inhalation of isoflurane. Phosphate
Buffered Saline (PBS) was purchased from Thermo Fisher Scientific
(Waltham, MA), and saline (0.9%) was made from sodium chloride
and Millipore water. Isoflurane was purchased from Phoenix
Pharmaceuticals, Inc. (St. Joseph, MO), and pharmaceutical-grade
DMSO was purchased from Sigma-Aldrich (St. Louis, MO). Medical-
grade oxygen was purchased from Praxair (Danbury, CT).
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplementary figures. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
chrischang@berkeley.edu
Animals. FVB-luc+ (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J) mice
were bred at UC Berkeley and were single- or group-housed on a
12:12 light−dark cycle at 22 °C with free access to food and water. All
studies were approved and performed according to the guidelines of
the Animal Care and Use Committee of the University of California,
Berkeley.
Comparison of in Situ Luciferin Formation and Luciferin in
Mice. Unshaven, male FVB-luc+ mice were anesthetized with
isoflurane and injected i.p. with D-cysteine (0.05 or 0.5 μmol, in 20
μL of PBS) or vehicle (20 μL of PBS). After 2 min, mice were injected
i.p. with HCBT (0.05 or 0.5 μmol in 50 μL of 1:1 DMSO:PBS) or
luciferin (0.05 μmol in 50 μL of 1:1 DMSO:PBS). Following
injections, mice were imaged with an IVIS Spectrum.
Determination of Signal Produced from Endogenous L-
Cysteine in Mice. Unshaven, male FVB-luc+ mice were anesthetized
with isoflurane and injected i.p. with D-cysteine (0.05 μmol, in 20 μL
of PBS) or vehicle (20 μL of PBS). After 2 min, mice were injected i.p.
with HCBT (0.05 μmol in 50 μL of 1:1 DMSO:PBS). Following
injections, mice were imaged with an IVIS Spectrum.
Exogenous H2O2 Experiments in Mice. Unshaven, male FVB-
luc+ mice were anesthetized with isoflurane and injected i.p. with a
mixture of PCL-2 and D-cysteine (0.05 μmol each, in 50 μL of 1:1
DMSO:PBS), followed immediately by an i.p. injection of H2O2 (0−
4.5 μmol in 100 μL of PBS). Control mice were injected i.p. with a
mixture of HCBT and D-cysteine (0.01 μmol each, in 50 μL of 1:1
DMSO:PBS) immediately prior to H2O2 (4.5 μmol in 100 μL of PBS).
Following injections, mice were imaged with an IVIS Spectrum.
Antioxidant Experiments in Mice. Unshaven, male FVB-luc+
mice were anesthetized with isoflurane and injected i.p. with NAC (10
mg/kg in 25 μL of PBS, pH 7−8) or PBS (25 μL). After 2 min, the
mice were injected i.p. with a mixture of PCL-2 and D-cysteine (0.05
μmol each, in 50 μL of 1:1 DMSO:PBS) immediately prior to i.p.
injection of H2O2 (1.5 μmol in 75 μL of PBS). Firefly luciferin control
mice were injected i.p. with NAC (10 mg/kg in 100 μL of PBS, pH 7−
8) or PBS (100 μL) immediately following i.p. injection of a mixture of
HCBT and D-cysteine (0.01 μmol each, in 50 μL of 1:1 DMSO:PBS).
Following injections, mice were imaged with an IVIS Spectrum.
Lipopolysaccharide Inflammation Model. Unshaven, female
FVB-luc+ mice, aged 2−5 months, were anesthetized with isoflurane
and injected i.p. with LPS (3 mg/kg in 50 μL of saline) or saline (50
μL). For studies with PCL-2 alone, mice were injected i.p. with either
apocynin (10 mg/kg in 20 μL of DMSO) or DMSO (20 μL) 6 h after
LPS injection. Two minutes after this injection, mice were injected i.p.
with a mixture of PCL-2 and D-cysteine (0.05 μmol each, in 50 μL of
1:1 DMSO:PBS) and imaged. For studies with IETDC alone, mice
were injected i.p. with either z-VD(OMe)-OPh (1 μmol in 20 μL of
DMSO) or DMSO (20 μL) 5.5 h after LPS injection. Thirty minutes
after z-VD(OMe)-OPh or vehicle injection, mice were injected i.p.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institute of General Medical Sciences
(NIH GM 79465 to C.J.C. and NIH GM 058867 to C.R.B.)
and the Packard Foundation (C.J.C.) for funding this work.
C.J.C. and C.R.B. are Investigators with the Howard Hughes
Medical Institute. We thank David King (UC Berkeley) for
synthesis of the IETDC peptides and help with purification of
the peptides. We thank Ann Fischer (UC Berkeley) for
culturing and plating of PC3M-luc cells. We thank Prof. Elena
Dubikovskaya for preliminary discussions and experiments that
led to this study.
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