An Activity-Based Sensing Approach for the Detection of Cyclooxygenase-2 in Live Cells

Article abstract of DOI:10.1002/anie.201914845

Cyclooxygenase-2 (COX-2) overexpression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX-2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity-based sensing approach for monitoring COX-2 within live cells with confocal microscopy and flow cytometry. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX-2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen-dependent changes in COX-2 activity that are independent of protein expression within live macrophage cells.

Full text of DOI:10.1002/anie.201914845

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: An Activity-based Sensing Approach for the Detection of
Cyclooxygenase-2 in Live Cells
Authors: Anuj K. Yadav, Christopher J. Reinhardt, Andres S. Arango,
Hannah C. Huff, Liang Dong, Michael G. Malkowski, Aditi
Das, Emad Tajkhorshid, and Jefferson Chan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914845
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10.1002/anie.201914845
Angewandte Chemie International Edition
RESEARCH ARTICLE
An Activity-based Sensing Approach for the Detection of
Cyclooxygenase-2 in Live Cells
Anuj K. Yadav,+[a] Christopher J. Reinhardt,+[a] Andres S. Arango,[b] Hannah C. Huff,[a] Liang Dong,[c]
Michael G. Malkowski,[c] Aditi Das,[d] Emad Tajkhorshid,[b] and Jefferson Chan[a]*
difference between the two isoforms is their expression profile:
COX-1 is constitutively expressed in most cell types, whereas
COX-2 expression can either be constitutive or induced for rapid
prostaglandin production depending on the tissue type.[2,4,9–12]
COX overexpression represents a prominent phenotype in
Abstract: Cyclooxygenase-2 (COX-2) overexpression is prominent in
inflammatory diseases, neurodegenerative disorders, and cancer.
Directly monitoring COX-2 activity within its native environment poses
an exciting approach to account for and illuminate the effect of the
local environments on protein activity. Herein, we report the
development of CoxFluor, the first activity-based sensing approach
for monitoring COX-2 within live cells with confocal microscopy and
flow cytometry. CoxFluor strategically links a natural substrate with a
dye precursor to engage both the cyclooxygenase and peroxidase
activities of COX-2. This catalyzes the release of resorufin and the
natural product, as supported by molecular dynamics and ensemble
docking. CoxFluor enabled the detection of oxygen-dependent
changes in COX-2 activity that are independent of protein expression
within live macrophage cells.
inflammation,[13,14]
neurodegenerative
disorders,[15]
and
cancer.[10,16,17] For example, high expression levels and cross-talk
with inducible nitric oxide synthase in cancer can correlate with
poor clinical outcomes through increased angiogenesis,
proliferation, and cell migration.[18–22] It is important to note that a
variety of factors beyond concentration can influence COX-2
activity (e.g., substrate concentration, allosteric regulators, and
post-translational modifications), especially in vivo.[23–27]
To date, only a limited number of strategies have been
introduced for detecting COX-2 in living systems. For example,
selective inhibitors of COX-2 have been radiolabeled[28–34] or
appended to dyes[35,36] for PET/SPECT or fluorescence imaging,
respectively. These imaging agents report on relative COX-2
expression profiles; however, they remain in a constant ‘on’ state,
regardless of whether they are bound to COX-2. More recently,
activatable fluorescent inhibitors have been reported that afford
an “off-on” response, where protein binding sequesters the
inhibitor away from the fluorophore, disrupts the inhibitor-
mediated fluorescence quenching, and renders the protein-small-
Introduction
Cyclooxygenase (COX, E.C. 1.14.99.1) is the key
biosynthetic enzyme that initiates the synthesis of prostaglandins
from their linear lipid precursor, arachidonic acid (AA).[1–3] After
production, the prostaglandins function as important lipid-based
mediators that regulate physiological processes, such as gastric
epithelial protection, hemostasis, and sodium metabolism.[4,5] At
higher concentrations the prostaglandins act as potent pro-
inflammatory compounds through the production of cytokines.[6,7]
These prostaglandins can be further elaborated by a variety of
tissue-specific isomerases and synthases to afford the entire
gambit of prostanoids (AA-derived prostaglandins).[5]
Two isoforms of human COX have been identified (COX-1
and -2), which display a high level of structural homology (~60%
sequence identity).[8] Both isoforms catalyze the rate-limiting step
for production of prostaglandin H2 (PGH2) through distinct
cyclooxygenase and peroxidase active sites. The major
molecule adduct fluorescent.[37,38]
Despite these notable
improvements, these technologies only report on whether COX-2
is present, but not whether the enzyme is catalytically active.
Activity-based sensing (ABS) represents
a powerful
detection strategy that relies on selective chemical reactivity. In
contrast to binding-based methods, ABS provides a direct readout
for the analyte or protein’s activity, rather than its concentration,
enabling a more complete depiction of its contribution within living
systems.[39,40] A variety of fluorescent probes have been reported
for the detection of enzyme activity. However most display broad
reactivity across a class of enzymes.[41] The development of
isoform-selective activity-based sensors remains challenging due
to the generally high structural similarities among isoforms. To
date, some examples exist; however, they have largely targeted
promiscuous enzymes involved in xenobiotic metabolism (e.g.,
cytochrome
P450,
monoamine
oxidase,
aldehyde
[a]
Dr. A. K. Yadav, C. J. Reinhardt, H. C. Huff, Prof. E. Tajkhorshid,
Prof. J. Chan
Department of Chemistry, Beckman Institute for Advanced
Science and Technology, University of Illinois at Urbana-
Champaign, Urbana, Illinois, 61801 United States
E-mail: jeffchan@illinois.edu
A. S. Arango, Prof. A. Das, Prof. E. Tajkhorshid
Center for Biophysics and Quantitative Biology, Department of
Biochemistry, University of Illinois at Urbana-Champaign, Urbana,
IL, 61801 United States
dehydrogenase).[42–46] To address these limitations, we
developed CoxFluor, the first isoform-selective, activity-based
fluorescent probe for COX-2. Isoform selectivity was achieved by
leveraging the subtle differences in the size and dynamics of the
cyclooxygenase active sites.[2,47] CoxFluor enabled the validation
of COX-2 inhibitors within in vitro assays, was successfully
applied for live-cell imaging and flow cytometry, and illustrated
oxygen-dependent COX-2 activity within live macrophage.
[b]
[c]
[d]
Dr. L. Dong, Prof. M. G. Malkowski
Department of Structural Biology, Jacobs School of Medicine and
Biomedical Sciences, University at Buffalo, Buffalo, NY, 14203,
United States
Results and Discussion
Prof. A. Das
CoxFluor consists of 3,7-dihydroxyphenoxazine (reduced
form of resorufin) linked to AA through a cleavable amide bond.
We proposed this design with the hypothesis that the lipid tail
could serve as a substrate for COX-2 in a manner similar to the
AA (Scheme 1a). After binding within the cyclooxygenase active
site, the probe could undergo hydrogen atom abstraction by the
Department of Comparative Biosciences
University of Illinois at Urbana-Champaign
Urbana, Illinois, 61801 United States
[+] These authors contributed equally to this work.
Supporting information for this article is given via a link at the end
of the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201914845
Angewandte Chemie International Edition
RESEARCH ARTICLE
Scheme 1. (a) Biosynthesis of PGH2 from AA by COX. (b) Proposed mechanism of COX-catalyzed resorufin production via CoxFluor-PGG2 intermediate oxidation
by Compound I (p cationic porphyrin radical) or Compound II (oxyferryl). The reaction between two CoxFluor-PGG2 intermediate radicals, dismutation, and hydrolysis
are omitted for simplicity.
With CoxFluor in hand, we first evaluated its response to
recombinant human COX-2. Treatment of CoxFluor (ΦF = 0.29 , ε
= 820 M-1cm-1 at 572 nm) with COX-2 resulted in the production
of resorufin (ΦF = 0.55, ε = 73,000 M-1cm-1 at 573 nm)[56] and PGG2
(confirmed by LC-HRMS, Figure S1) with a 41-fold fluorescent
turn-on response (Figure 1a).
enzyme’s Tyr385, followed by dioxygenation and cyclization to
afford the CoxFluor-PGG2 intermediate. Subsequent
translocation to the peroxidase active site and oxidation by
Compound I or II could yield an unstable oxygen-centered
resorufin radical followed by dismutation and amide hydrolysis to
release the fluorescent product and either PGG2 or PGH2 (after
reduction, Scheme 1b).[1,48–50] When proposing this structure, we
hypothesized that we could utilize the bulky dye to prevent COX-
1 binding and catalysis. This was based on previous reports that
indicate COX-1 is unable to accommodate large groups at the
carboxylate[2] and that this moiety provides critical interactions
with Arg120 for binding.[51–53] On the other hand, COX-2’s larger
substrate pocket and expanded substrate scope, including
amides, suggested that CoxFluor should be competent for
catalysis.[54]
To confirm that activation was a consequence of enzyme-
catalyzed oxidation rather than unbiased peroxidase activity, we
prepared a control compound, Ctrl-CoxFluor, where the AA lipid
was replaced with the saturated lipid tail (arachidic acid, Scheme
S1). Ctrl-CoxFluor proved to be unreactive under the same
reaction conditions (Figure 1b), strongly suggesting that the signal
enhancement observed for CoxFluor was due to enzymatic
activity. Under steady state conditions, CoxFluor underwent
oxidation to resorufin with a kcat of 19 min-1 and Km of 22 µM
(Figure 1c). The Km values are similar to those obtained for
arachidonyl ethanolamide (Km = 24 µM) and were within an order
of magnitude of AA (Km = 6 µM) and the measured rate for turn-
over is within 100-fold of the natural substrate cyclooxygenase
activity.[54] Moreover, CoxFluor’s fluorescence response was
inhibited by both indomethacin and celecoxib (Figure 1d). This
suggests that CoxFluor can be utilized to identify and/or evaluate
COX-2 inhibitors, overcoming key drawbacks in existing
methodologies that require radiolabeled compounds, purification
of intermediates or products, or coupled-enzyme systems.[57,58]
This also provides further support for the formation of a CoxFluor-
PGG2 intermediate because both inhibitors bind within the
cyclooxygenase active site of the protein.[59,60]
Scheme 2. Synthesis of CoxFluor from resazurin.
O
O
O
O
O
ONa
1) Zn, AcOH
O
O
N
H
N
O
2) Ac2O, DMAP
Acetone
Resazurin
63%
1
O
O
O
N
O
O
HO
O
N
OH
Arachidonyl-Cl
Na2CO3, KI
Na
O
O
CH3CN
MeOH
Isoform selectivity was evaluated by incubating CoxFluor
with COX-1 isolated from bovine vesicles, where a minimal
change in fluorescence was observed over the course of 4 h (1.3-
fold fluorescence enhancement, 5% of COX-2 response, Figure
S2). Off-target activation was also assessed against a panel of
enzymes that possess closely related activities (lipoxygenase,
peroxidase, catalase), could potentially cleave the amide
(esterase), or are capable of metabolizing AA analogs
(cytochrome P450s).[61] Even in the presence of 10-fold excess
enzyme, CoxFluor displayed good selectivity against all of the
tested enzymes (Figure 1e). Likewise, no undesirable activation
was observed when CoxFluor was incubated with various
biologically relevant reactive oxygen species, reactive nitrogen
species, or other cellular oxidants/reductants (Figure 1f and S3).
Moreover, no bleaching was observed when resorufin was treated
49%
2
68%
CoxFluor
The synthesis of CoxFluor began with the Zn-mediated
reduction of resazurin, followed by acetylation with acetic
anhydride to afford 1 in 63% yield over two steps.[55] This
intermediate was then coupled to arachidonyl chloride in the
presence of potassium iodide to afford 2 in 49% yield. Finally,
treatment of 2 with sodium methoxide (generated in situ)
facilitated acetyl deprotection and provided CoxFluor in 68% yield
(overall 21% over 4 steps, Scheme 2).
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10.1002/anie.201914845
Angewandte Chemie International Edition
RESEARCH ARTICLE
with the same panel of reactive oxygen and nitrogen species
(Figure S3). Importantly, incubation of CoxFluor with COX-2 in the
presence of glutathione (GSH, 1 mM) maintained significant
COX-2-specific fluorescence enhancement, indicating the
potential for use for live-cell imaging (Figure S4). This is in
contrast to Amplex® Red-based assays, which display cross-
reactivity with GSH in the presence of peroxidases[62] or direct
quenching of the radical intermediate.[50]
2 (PDB 5KIR)[64] were selected, ligands were removed and the
holoenzyme was simulated in explicit water over a period of 200
ns. Importantly, no global differences were observed for the
peptide backbone across
a panel of COX-1 and COX-2
structures, even in the presence of ligands and substrate
(www.rcsb.org, all root-mean-standard-deviation < 2.5 Å, Table
S1-2 and Figure S5).[65] Next, CoxFluor was docked into each
pose along the simulation trajectory to probe the most probable
binding modes (based on the predicted binding score),[66] where
clear differences in the number and area available for binding
were observed both overall and within the cyclooxygenase active
site of COX-2 as compared to COX-1 (Figure 2a-c and S6). This
finding is consistent with the difference in solvent accessible
surfaces harbored within each cyclooxygenase active site.[2]
CoxFluor also docks in a similar location to the reported
indomethacin fluorescent inhibitors (Figure S7),[36,67] further
supporting the proposed cyclooxyenase-dependent release of
resorufin (Scheme 1b).
a
b
50
40
30
20
10
0
35
30
25
20
15
10
5
0
a
b
550 575 600 625 650 675 700
r
r
Wavelength (nm)
CoxFluo
Ctrl-CoxFluo
c
d
25
20
15
10
5
20
15
10
5
kcat = 19 ± 1 min-1
Km = 22 ± 3 μM
c
d
80
60
40
20
0
COX-1
COX-2
0
0
2
X-
n
b
0
20
40
60
O
[CoxFluor] (μM)
C
Celecoxi
Indomethaci
e
f
30
25
20
15
10
5
30
25
20
15
10
5
-10 -8
-6
-4
-2
0
Binding Affinity (kcal/mol)
Figure 2. Molecular dynamics and ensemble docking studies of (a) COX-1 and
(b) COX-2 with CoxFluor (green). Initial protein structures were prepared from
PDB 5U6X and 5KIR and crystal bound AA (orange) is superimposed for
comparison from PDB 1DIY and 3HS5, respectively. Structures display an
overlay of all CoxFluor docked poses with scoring less than or equal to -7
kcal/mol. The heme from the simulation is represented as a ball and stick model
to distinguish the cyclooxygenase and peroxidase active sites. (c) Percent of
CoxFluor poses within the docking score binding affinities. (d) Competent
binding of CoxFluor within the cyclooxygenase active site. Tyr385 (yellow) is
oriented for hydrogen atom extraction from either CoxFluor (green) or AA
(orange, PDB 3HS5).
0
0
-
-
-
-
-
2
O
HN
ONO
2
H
2
2
3
NO
O
X-
O
O
O
H 2
tBuOO
Cl
O
RLMHRP
MPO
NO NO
C
5-LOX
COXC-1OX-2
MAO-A
EsteCraasteaClaYsPe2J2
Figure 1. (a) Relative fluorescence for CoxFluor (10 µM) incubated with COX-
2 (250 nM) and hemin (1 µM) over the course of 4 h. (b) Relative fluorescent
intensity of CoxFluor and Ctrl-CoxFluor after incubation with COX-2 (250 nM)
and hemin (1 µM) for 4 h. (c) Michaelis-Menten kinetics for COX-2-catalyzed
(50 nM) release of resorufin in the presence of hemin (200 nM). (d) Inhibition of
COX-2 (250 nM) and hemin (1 µM) by indomethacin (10 µM) and celecoxib (10
µM). (e) Relative fluorescence for CoxFluor incubated COX-2 (0.101 U), a panel
of enzymes (10-fold excess) or (f) panel of reactive oxygen/nitrogen species
(500 µM, 50 equiv.). All experiments were conducted at room temperature in
100 mM Tris HCl buffer (pH 8.0) except MPO, and 5-LOX (additional information
in the Supporting Information). All data is reported as the mean ± standard
deviation (n = 3) for parts b, d, e and f or the error from fitting for part c. Parts
a/c and b/d-f were performed according to the fluorimeter and plate reader
assays, respectively.
To begin to interrogate the sequence of events, CoxFluor
and the CoxFluor-PGG2 intermediate were docked within COX-2.
Consistent with the proposed mechanism, CoxFluor binds
significantly at the cyclooxygenase active site (66/597 poses at -
7 kcal/mol cutoff). On the other hand, the CoxFluor-PGG2
intermediate displayed increased binding at the peroxidase active
site for oxidation by either Compound I or Compound II (356/368
poses at -7 kcal/mol cutoff). This increased selectivity for the
CoxFluor-PGG2 intermediate at the peroxidase active site is
consistent over all spontaneous interactions (Figure S8). These
results are consistent with peroxidase oxidation[68] of CoxFluor-
PGG2 in a similar manner to Amplex® Red[50] and 2,2′-azinobis(3-
ethylbenzothiazolinesulfonic acid oxidation by horseradish
peroxidase.[49,69] Finally, to confirm that CoxFluor can adopt the
To further investigate the mechanism of CoxFluor activation
by COX-2, we employed molecular dynamics (MD) and ensemble
docking. Previous structures of COX-1 (PDB 5U6X)[63] and COX-
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10.1002/anie.201914845
Angewandte Chemie International Edition
RESEARCH ARTICLE
requisite conformation for oxidation by the active site Tyr385,
CoxFluor was docked within the cyclooxygenase active site and
compared to crystal bound AA (PDB 3HS5).[70] Structural
comparisons indicate that CoxFluor can bind within the
cyclooxygenase active site with proper orientation for oxidation
(Figure 2d). Similar poses are also observed within the larger box
that contains both the cyclooxygenase and peroxidase active
sites. Together, these results suggest that CoxFluor binds within
the substrate binding pocket or adjacent solvent-exposed sites to
undergo oxidation and formation of the CoxFluor-PGG2
intermediate. After dissociation from the active site, the
intermediate displays increased binding to the peroxidase active
site for oxidation by Compound I or Compound II and subsequent
release of resorufin.
indomethacin it was possible to clearly identify COX-2 activity
across activation states (M0 versus M1, Figure S15). Finally, the
ability of CoxFluor to detect activity changes without GSH
depletion was confirmed with flow cytometry, under similar
conditions, where a 1.2-fold increase in activity was observed
after 19 h LPS-activation relative to 4 h LPS-stimulated cells
(Figure S16).
a
b
Prior to performing cellular experiments, we evaluated
CoxFluor’s stability and biocompatibility. Negligible fluorescence
response was observed after
8 h of incubation at room
temperature or 37 °C (Figure S9) and typical staining conditions
yielded minimal toxicity in HEK 293T and RAW 264.7
macrophage cells (Figure S10). Next, we generated a transiently
transfected HEK 293T cell line over-expressing human COX-2.
GSH depletion was performed with N-ethylmaleimide (NEM)[71]
prior to staining because GSH can generate[62] or quench radical
intermediates of peroxidase-based probes, thereby complicating
the interpretation of results.[50,72] Moreover, past work has
demonstrated that COX-2 inhibitors (e.g., celecoxib analogs and
indomethacin) can alter GSH levels,[73–76] which is consistent with
our initial experimental observations (Figure S11). After
incubation with CoxFluor for 3 h we observed a 1.2-fold
fluorescence increase for transfected cells compared to the
control, and no fluorescence enhancement was observed upon
treatment with indomethacin (Figure S12).
3
2
1
0
c
d
**
ns
Control
LPS
Indo.
min
max
Next, we applied CoxFluor for the detection and imaging of
endogenous COX-2 activity within RAW 264.7 murine
macrophage. As a key player in inflammatory and immune
responses, macrophages undergo phenotypic changes to pro-
inflammatory or anti-inflammatory states upon stimulation.[77]
Because COX-2 activity is the rate-limiting step in prostaglandin
biosynthesis (pro-inflammatory mediators), COX-2 expression
and subsequent prostaglandin production can be measured in
well-established lipopolysaccharide (LPS)-induced inflammation
models as a biomarker for inflammation.[3,78–83] To date, these
studies typically rely on mRNA quantification, western blot
analysis, or downstream product quantification (e.g., ELISA
assays) rather than direct quantification of COX-2 activity. This is
due to the lack of isoform selectivity in current COX assays. First,
we measured the effect of LPS stimulation time on COX-2’s
specific activity in RAW 264.7 macrophage lysates. Variations
due to GSH fluctuations were again minimized using NEM, which
we and others have shown does not affect COX activity (Figure
S13).[84] Within the first 4 h of activation only a modest 2.4-fold
increase in COX-2 activity was observed. Over the next several
hours, the activity increased in a linear fashion to a maximum of
17.6-fold at 19 h (Figure S14). These results are similar to
previous reports of prostaglandin biosynthesis within both
murine[82] and human[81] macrophage cells under LPS-stimulated
conditions. The activity differences were then confirmed within
live cells using both confocal microscopy (Figure 3) and flow
cytometry (Figure S15). Rather than using NEM, we pre-treated
with buthionine sulfoximine (BSO), a g-glutamylcysteine synthase
inhibitor, because it is a less cytotoxic alternative.[85,86] Under
these conditions we observed a 1.2-fold fluorescent enhancement
for LPS-stimulated cells relative to the control using confocal
microscopy and a 1.6-fold increase in median fluorescence for
cells activated for 19 h as compared to 4 h with flow cytometry
(Figure 3 and S15). By monitoring the ratio of fluorescence from
CoxFluor stained cells to CoxFluor stained cells treated with
Figure 3. Confocal imaging of COX-2 activity in (a) control, (b) LPS-stimulated
(1 µg/mL), and (c) indomethacin-treated (10 µM), LPS stimulated RAW 264.7
cells after 4 h incubation with CoxFluor (10 µM) at 37 ºC following treatment with
BSO (began 19 h prior to staining, 200 µM). Scale bar (white) represents 10 µm.
(d) Quantified data. Values are reported as the mean ± standard deviation (n =
3). Statistical analysis was performed using one-way ANOVA (α = 0.05). **, p <
0.01.
Finally, we were interested in determining whether COX-2
activity can be regulated beyond the protein level. We
hypothesized that oxygen concentrations may influence COX-2
activity because oxygen serves as a substrate, but can also bind
ferric heme prosthetic groups to yield an off-cycle resting state.[87]
The effect of oxygen on COX-2 activity was evaluated in live RAW
264.7 macrophage cells after a 19 h activation with LPS. The cells
were then stained under normoxic (~21%) or hypoxic conditions
(prepared using AneroPack® for < 0.1% oxygen in a sealed
container) and the fluorescent response was monitored with flow
cytometry. Interestingly, higher activity was observed under
hypoxic conditions, as indicated by a substantial shift in the
median fluorescence (Figure 4a-c). Moreover, this increase in
fluorescence was COX-2-dependent, where treatment with
indomethacin resulted in a 12% decrease in the ratio of median
fluorescence for hypoxic to normoxic M1 macrophages (Figure
4d). To confirm the result and evaluate the dose-dependency of
oxygen, we repeated the experiment under a 1 or 0.1% oxygen
atmosphere. Here we observe an oxygen-dependent decrease in
COX-2 activity (Figure 4d-e) that is independent of protein
expression levels (Figure 4f). This provides strong evidence that
the local cellular environment plays a critical role in the regulation
of COX-2 activity. This finding also emphasizes the importance of
measuring activity, in addition to transcriptional and translational
levels. To the best of our knowledge, this represents the first
example when an ABS approach has revealed activity differences
that result from regulation beyond protein expression levels.
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10.1002/anie.201914845
Angewandte Chemie International Edition
RESEARCH ARTICLE
activity can be regulated by oxygen availability within live cells.
Taken together, these results suggest that CoxFluor will be a
powerful tool for evaluating new therapeutics and for studying
COX-2 in live cells.
Acknowledgements
The authors acknowledge the National Institutes of Health for
support (R35GM133581 to J.C.; R01GM115386 to M.G.M.;
P41GM104601 and R01GM123455 to E.T.; R01GM1155884 and
R03DA04236502 to A.D.). This work was supported in part by the
Alfred P. Sloan Fellowship (FG-2017-8964 to J.C.) and by the
American Heart Association Scientist Development Grant
(15SDG25760064 to A.D). C.J.R. acknowledges the Chemistry-
Biology Interface Training Grant (T32GM070421) and the
Seemon Pines Graduate Fellowship for support. Simulations
were performed using the National Science Foundation’s
computing resources through an XSEDE grant (TMGCA06N060
to E.T.) and PRAC allocation grant (ACI1713784 to E.T.). Major
funding for the 500 MHz Bruker CryoProbeTM was provided by
the Roy J. Carver Charitable Trust (Muscatine, Iowa; Grant #15-
4521) to the School of Chemical Sciences NMR Lab. The Q-TOF
Ultima mass spectrometer was purchased in part with a grant
from the National Science Foundation, Division of Biological
Infrastructure (DBI-0100085). We acknowledge Prof. Elvira de
Mejia (Food Science and Human Nutrition, UIUC) for providing
RAW 264.7 macrophage cells. We acknowledge the Core
Facilities at the Carl R. Woese Institute for Genomic Biology for
access to the Zeiss LSM 700 Confocal Microscope. Additionally,
we acknowledge the Roy J. Carver Biotechnology Center at UIUC
and Dr. Barbara Pilas for access to the BD LSR Fortessa Flow
Cytometry Analyzer and for constructive discussions about
instrumentation. We also thank Prof. Robert Gennis (Department
of Biochemistry, UIUC) for access to the Strathkelvin oxygen
electrode, Prof. H. Rex Gaskins (Department of Animal Sciences
and Pathobiology, UIUC) and Emily (Jee-Wei) Chen (Department
of Chemical and Biomolecular Engineering, UIUC) for access and
assistance with the BioSpherix C-Chamber hypoxic incubator
equipped with the corresponding OxyCycler C42 sub-chamber
controller, and Daryl Meling and Amogh Kambalyal for the
procurement and dissection of the bovine vesicles.
Figure 4. Contour plots of flow cytometry data from live LPS-activated RAW
264.7 macrophage cells stained with CoxFluor (10 µM) under (a) normoxic and
(b) hypoxic conditions. (c) Quantified median fluorescence for hypoxic and
normoxic cells. (d) Effect of indomethacin (20 µM) treatment on the ratio of
hypoxic to normoxic median fluorescence. (e) Median fluorescence of RAW
264.7 macrophage cells stained under 0.1% (red), 1.0% (grey), or 21% (blue)
oxygen concentrations. (f) ELISA quantification of COX-2 protein expression
levels in 25 µg/mL protein lysates. Values are reported as the mean ±
propagated standard deviation (n
= 6 for flow cytometry data; n = 3
indomethacin inhibition and ELISA data). All staining was performed following
treatment with BSO (began 2 h prior to staining, 200 µM) and oxygen dependent
flow cytometry data was replicated on two separate days. Statistical analysis
was performed with two-tail Student’s t test for c, one-tail Student’s t test for d,
or one-way ANOVA for e (α = 0.05). *, p < 0.05, ****, p < 0.0001.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: activity-based sensing • bioimaging • fluorescent
probes • isoform selectivity • molecular dynamics and ensemble
docking
Conclusion
It is critical to develop methods for studying the influence of
the local environment on COX-2’s enzymatic activity. While it is
reasonable to postulate that substrate availability should affect
reaction rates, the outcome is particularly difficult to predict within
living systems. We turned to an ABS approach to overcome this
key challenge. CoxFluor represents the first isoform-selective
probe for measuring COX-2 activity. Strategic utilization of the
bulky resorufin precursor and AA tail provided CoxFluor with the
requisite selectively for interrogating COX-2 biology in complex
mixtures (e.g., in the presence of COX-1), where current inhibitors
display imperfect selectivity.[88] This isoform selectivity enables
researchers to directly study COX-2 within its native environment,
while accounting for factors beyond transcription and
translation.[89] Along these lines, we demonstrate that COX-2
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RESEARCH ARTICLE
RESEARCH ARTICLE
Activity-based sensing of
Cyclooxygnease-2: An
Anuj K. Yadav, + Christopher J.
Reinhardt, + Andres S. Arango,
Hannah C. Huff, Liang Dong,
Michael G. Malkowski, Aditi Das,
Emad Tajhorshid, Jefferson
Chan*
isoform-selective fluorogenic
probe reports on enzymatic
activity within live cells. Two-
step activation facilitates the
release of the natural product
and resorufin for confocal
imaging and flow cytometry.
CoxFluor indicates that COX-2
activity can be regulated by
oxygen without a change in
protein expression level.
Page No. – Page No.
An Activity-based Sensing
Approach for the Detection of
Cyclooxygenase-2 in Live Cells
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