An activity-based imaging probe for the integral membrane hydrolase KIAA1363

Article abstract of DOI:10.1002/anie.201107236

Profiling enzyme activities: A selective, activity-based imaging probe is described for the integral membrane hydrolase KIAA1363 and used to determine the subcellular localization and half-life of this enzyme in human cancer cells (see picture). Copyright

Full text of DOI:10.1002/anie.201107236

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DOI: 10.1002/anie.201107236
Fluorescent Probes
An Activity-Based Imaging Probe for the Integral Membrane
Hydrolase KIAA1363**
Jae Won Chang, Raymond E. Moellering, and Benjamin F. Cravatt*
Activity-based protein profiling (ABPP) is a chemical pro-
teomic platform for characterizing enzyme activities in native
biological systems.[1–3] Original small-molecule probes for
ABPP were designed to target large numbers of enzymes that
share mechanistic and/or structural features. These efforts
have yieled activity-based probes for many enzyme classes,
including serine[4,5] and cysteine[6] hydrolases, oxidoreduc-
tases,[7] metalloproteases,[8] histone deacetylases,[9] kinases,[10]
and glycosidases.[11,12] ABPP has been applied to discover
enzyme activities that are deregulated in biological processes
such as cancer[13] and infectious disease.[14] Configuring ABPP
to operate in a competitive mode has further enabled the
development of selective inhibitors to probe the function of
disease-relevant enzymes in cell and animal models.[5,6,15] As
biological studies using ABPP have evolved, the need for
target-selective activity-based probes has also become appar-
ent. The specificity of such probes opens up new biological
applications, including direct spatial and temporal visual-
ization of active enzymes in cells and tissues.[16,17] While
attractive in principle, the development of protein-selective,
activity-based imaging probes poses substantial technical
challenges. Such a probe should ideally possess several
features, including high selectivity for a single enzyme
target, a reporter tag for imaging the probe-labeled enzyme,
and suitable cell permeability and pharmacokinetic proper-
ties for in vivo studies. These objectives have, so far, been
realized for only a handful of probes that label proteolytic
enzymes[18] and whether they can be achieved for probes that
target additional types of enzymes remains unknown.
In cancer cells, KIAA1363 regulates a set of pro-tumorigenic
ether lipids and disruption of this metabolic pathway with the
KIAA1363 inhibitor JW480 impairs cancer cell migration and
tumor growth in vivo.[19] Here we have asked whether
carbamate inhibitors could be converted into selective
chemical probes for spatial and temporal imaging of
KIAA1363 activity in cancer cells. There is a particular
need for activity-based imaging probes for KIAA1363, since
this enzyme is subject to extensive and variable post-trans-
lational modification (primarily glycosylation),[21] which has,
so far, impeded efforts to develop antibodies for immuno-
fluorescence imaging. Our established structure-activity rela-
tionship[19] suggested that the naphthyl-containing carbamoy-
lating arm of JW480 could potentially be replaced with a
hydrophobic fluorophore group without substantial losses in
potency or selectivity for KIAA1363 (Figure 1a).
Of the many types of fluorophores that could be
incorporated into a KIAA1363-selective probe, we chose
the BODIPY class owing to its similar overall size and
hydrophobicity compared to the naphthyl moiety in JW480
(Figure 1a). In the resulting molecule, JW576, the BODIPY
fluorophore was appended to the parent carbamate structure
through a alkyl amide linker (Figure 1a and Scheme S1 in the
Supporting Information). Excitation and emission spectra of
JW576 revealed maxima at 505 and 512 nm, respectively
(Figure 1b). These results indicated that the embedded
fluorophore retains the spectral properties of the parent
BODIPY compound and should be suitable for fluorescence
detection of KIAA1363 and any other potential JW576-
reactive proteins.
We first evaluated whether JW576 specifically targeted
KIAA1363 by performing competitive ABPP with the serine
hydrolase-directed probe fluorophosphonate-rhodamine (FP-
Rh).[23] PC3 cancer cells, which have high endogenous
KIAA1363 levels,[19] were treated with JW576 in situ (0.01–
25 mm for 4 h), after which cells were homogenized, treated
with FP-Rh (1 mm), and analyzed by gel-based ABPP. JW576
was found to selectively inhibit both of the 40–45 kDa
glycoforms of KIAA1363 with a half maximal inhibitory
concentration (IC50) value of (0.34 Æ 0.15) mm (Figure 1c).
Re-scanning of the gel on the BODIPY fluorescence channel
confirmed that JW576 inhibition of FP-Rh labeling of
KIAA1363 was paralleled by the appearance of a JW576–
KIAA1363 covalent adduct (Figure 1c). Importantly, scan-
ning for BODIPY fluorescence also permitted detection of
any other JW576–protein adducts, which were only observed
at trace levels and at high concentrations of JW576 (ꢀ 5 mm)
above the IC50 value for KIAA1363 labeling (Figure 1c).
Similar profiles were observed for JW576 in PC3 cell lysates
and in other cancer cell lines (see Figure S1 in the Supporting
We recently used competitive ABPP to develop potent
and selective covalent carbamate inhibitors for the integral
membrane serine hydrolase KIAA1363[19] (also known as
AADACL1 or NCEH1), which is highly expressed in
aggressive human cancer cell lines and primary tumors.[19–22]
[*] J. W. Chang,[+] Dr. R. E. Moellering,[+] Prof. Dr. B. F. Cravatt
Department of Chemical Physiology, The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037(USA)
E-mail: cravatt@scripps.edu
[+] These authors contributed equally to this work.
[**] We would like to thank A. Adibekian and the Cravatt lab for insightful
discussions and W. Kiosses and the TSRI Microscopy Core for
technical assistance. This research was supported by the California
Institute for Regenerative Medicine (predoctoral fellowship to
J.W.C.), the American Association for Cancer Research (Centennial
predoctoral fellowship to R.E.M.), the Damon Runyon Cancer
Research Foundation (HHMI postdoctoral fellowship to R.E.M.),
the US National Institutes of Health (grant number CA087660), and
the Skaggs Institute for Chemical Biology.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107236.
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that JW576 serves as a potent and selective fluorescent
inhibitor for KIAA1363 in cancer cells.
We next assessed whether JW576 could be used to image
KIAA1363 activity in cancer cells by confocal fluorescence
microscopy. PC3 cells were treated with JW576 (0.5 mm) for
0.5–4 h and imaged on BODIPYand DAPI channels to detect
retained JW576 signals and nuclear DNA, respectively.
Significant, time-dependent increases in BODIPY fluores-
cence were observed throughout the cancer cell body (Fig-
ure 2a, top), and these signals were blocked by co-treatment
with 10X JW480 inhibitor (Figure 2a, bottom). These results
indicated that the observed BODIPY fluorescence signals
reflect specific labeling of KIAA1363 by JW576, as opposed
to nonspecific retention of the compound. Also supporting
this conclusion, gel-based ABPP showed specific, time-
dependent, and JW480-sensitive labeling of KIAA1363 by
JW576 (Figure 2b). With the parameters for fluorescence
imaging established, we performed confocal microscopy
experiments in three aggressive human cancer cell lines
from distinct tumors of origin—prostate (PC3), breast
(231MFP), and ovarian (SKOV3)—all of which are known
to express high levels of KIAA1363.[13,19] In each case, strong,
JW480-sensitive intracellular staining was observed for
JW576-treated cells (Figure 2c). Similarly strong intracellular
staining was detected in COS7 cells transfected with a
KIAA1363 cDNA, but not in mock-transfected COS7 cells
(Figure 2d). These results establish the utility of JW576 as a
fluorescent imaging probe for direct visualization of active
KIAA1363 in cells and indicate that this enzyme is localized
predominantly to intracellular membrane compartments in
cancer cells.
We sought to determine the subcellular membrane
structure(s) that contain KIAA1363 activity by treating
cancer cells with JW576 (0.1 mm, 2 h) and then counterstain-
ing for markers of various subcellular organelles, including
the nucleus (DAPI), plasma membrane (wheat germ agglu-
tinin, WGA), and the endoplasmic reticulum (ER-tracker).
Confocal imaging of JW576-treated cells on wavelengths
specific to each marker revealed substantial colocalization of
KIAA1363 activity with the ER, and very little signal overlap
in the nucleus or at the cell surface (Figure 3a,b). Beyond
these three measurable locations, punctate patterns of JW576
staining were also observed on intracellular membranous
structures non-overlapping with the ER (Figure 3b, arrows),
indicative of some localization of KIAA1363 to other
organelles (possibly endosomal compartments). In an effort
to more completely describe the sub-cellular localization of
KIAA1363 activity, we performed quantitative co-localiza-
tion analysis. After gating each fluorescence channel to
exclude the autofluorescent background, we were able to
identify the locations in the cell where significant overlap
between two fluorescence channels occurred (Figure 3c).
Quantifying the percentage of each fluorescent signal that
overlapped with another marker confirmed substantial
( ꢁ 95%) localization of the KIAA1363 signal to the ER. In
contrast, the KIAA1363 activity showed minimal overlap
with the WGA or DAPI signals (Figure 3d, upper panel),
which also did not, as expected, overlap with each other
(Figure 3d, lower panel). These experiments have thus
Figure 1. Development of a fluorescent activity-based probe that selec-
tively targets the serine hydrolase KIAA1363. a) Design of JW576, an
activity-based probe that selectively targets KIAA1363. b) Excitation
and emission spectra of JW576. c) Competitive ABPP profiling of PC3
cells in situ. d) Time-course of KIAA1363 labeling by JW576 (1 mm) in
PC3 cells. e) Labeling of recombinant KIAA1363 by JW576. Mock- or
KIAA1363-transfected COS7 cells were treated with JW576 (0.1 mm)
with or without JW480 competitor (1 mm) for 2 h. f) Inhibition of 2-
AcMAGE hydrolysis activity of KIAA1363 in PC3 and SKOV3 cells
treated in situ with JW576 (MW=molecular weight).
Information). KIAA1363 inhibition and labeling by JW576
(1 mm) were also found to be time-dependent, with near-
complete target modification and negligible off-target inter-
actions observed within an hour in living PC3 cells (Fig-
ure 1d). We also confirmed specific labeling in COS7 cells
transfected with either a mock- or KIAA1363-expressing
plasmid, where JW576-labeled KIAA1363 signals were
abrogated by pre-treatment with JW480 (Figure 1e). Finally,
we found that JW576 blocked KIAA1363 activity in cancer
cells using a C16:0 2-acetyl MAGE substrate hydrolysis assay
(Figure 1 f and Figure S2 in the Supporting Information),
which provided similar in situ IC50 values (0.15–0.17 mm) as
competitive ABPP assays. Collectively, these data confirmed
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on the BODIPY channel revealed significant label-
ing of KIAA1363 at all tested doses (Figure 5a).
The rate of in vivo labeling of KIAA1363 by JW576
was fast, with near-maximal signals being observed
as early as 30 min post-dosing (Figure 5b). JW576
maintained good selectivity for KIAA1363 in vivo,
with only a single, faint additional probe target
(70 kDa protein) being detected in the heart
proteome (Figure 4a,b). We confirmed that the
40–45 kDa JW576-labeled protein doublet corre-
sponded to KIAA1363 by repeating in vivo treat-
ments in wild-type (WT) and KIAA1363 knockout
(KO) mice (Figure 5c). We also analyzed these
KIAA1363-WT and KO heart proteomes by com-
petitive ABPP with FP-Rh, which revealed sub-
stantial reductions in KIAA1363 activity in WT
mice treated with JW576 and complete absence of
KIAA1363 signals in KO mice (Figure 5c). These
competitive ABPP gels also provided evidence that
the 70 kDa protein labeled by JW576 was itself a
serine hydrolase (Figure 5c), likely corresponding
to the blood carboxylesterase ES1, which is a known
off-target of JW480,[19] the parent carbamate inhib-
itor from which JW576 was derived. Overall, these
Figure 2. Imaging of KIAA1363 activity in cancer cells treated with JW576. a) Con-
focal imaging of JW576-treated PC3 cells. b) Gel analysis of PC3 cells treated with
JW576 in the presence or absence of 10X JW480. c) Confocal imaging of prostate
(PC3), breast (231 MFP) and ovarian (SKOV3) cancer cells treated with
JW576Æ10X JW480 competitor for 2 h. d) JW576 treatment of mock- or KIAA1363-
transfected COS7 cells in the presence or absence of 10X JW480 for
2 h. Arrows in (b) indicate labeled-KIAA1363. Scale bars=5 mm.
identified the ER as the principle subcellular compartment
where KIAA1363 resides in cancer cells. It would be
interesting to determine, in future studies, if the distribution
of KIAA1363 in cancer cells is dynamically regulated by
signaling pathways or other cellular events, and JW576 should
offer a suitable imaging probe for these types of studies.
In addition to spatial mapping of active KIAA1363 in
cancer cells, we wondered whether JW576 could be used for
temporal tracking of the cellular turnover of KIAA1363. For
this study, we followed the general protocols for traditional
pulse-chase experiments, but used JW576 treatment in place
of metabolic incorporation of radiolabeled amino acids. Two
different human cancer cell lines (SKOV3 and DU145) were
pulse-treated with JW576 (5 mm) for 10 min, washed with
fresh media, and then incubated with media containing an
excess of JW480 to quench any unreacted KIAA1363. Cell
proteomes were then harvested at the indicated times over
48 h and KIAA1363 activity levels measured by gel-based
ABPP (Figure 4a,c). Time-dependent reductions in
KIAA1363 signals were observed in both cell lines and fitting
the quantified band intensities to an exponential decay model
provided half-life estimates of 14.2 and 22.9 h for KIAA1363
in SKOV3 and DU145 cells, respectively (Figure 4b,d).
An ideal target-selective, activity-based probe would not
only allow for visualization of enzyme activity in cells, but also
in animal models. We tested whether JW576 could label
KIAA1363 in C57Bl/6J mice by treating animals with
escalating doses of the compound (5–40 mgkgÀ1, i.p. (intra-
peritoneal injection) for 4 h. Mice were then sacrificed and
proteomes from heart, a tissue that expresses high levels of
KIAA1363,[24] were analyzed by gel-based ABPP. Scanning
Figure 3. JW576 enables temporal tracking of active KIAA1363 in
cancer cells. a,b) Confluent SKOV3 cells were treated with JW576
(0.1 mm, 2 h). Subcellular localization of active KIAA1363 populations
was analyzed by staining either the plasma membrane with wheat-
germ agglutinin (a) or the endoplasmic reticulum (ER) with ER-tracker
Red (b) followed by confocal imaging. Images show each fluorescence
channel separately and the overlay of all three. White arrows in the
zoomed inlay of (b) highlight punctate JW576-labeled regions not
stained by ER-Tracker. c) Quantitative colocalization analysis of JW576
with the ER (top) and plasma membrane (bottom). Areas of significant
signal overlap are false-colored white. d) Quantification of JW576 (top)
and DAPI (bottom) colocalization with the indicated subcellular
markers. Scale bars=20 mm.
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target KIAA1363 (but still inhibit ES1, a common off-target
for carbamates[24]). Considering that KIAA1363 activity is
highly elevated in aggressive human cancer cells and primary
tumors,[19–22] we anticipate that JW576 should serve as a
valuable functional probe to answer questions about
KIAA1363 expression and localization during tumorigenesis,
tumor progression, and metastasis. More generally, our
studies have extended the use of target-selective fluorescent
activity-based probes beyond cysteine proteases[16,17] to
include an enzyme from the serine hydrolase class. Consid-
ering the recent emergence of selective, covalent inhibitors
for many other serine hydrolases,[15,24] some of which are also
carbamates,[24] we suspect that the strategy described herein
should prove useful for developing activity-based imaging
probes for several enzymes from this class.
Figure 4. Temporal tracking of KIAA1363 turnover with JW576.
a,b) KIAA1363 protein half-life determination in SKOV3 cancer cells
was determined by pulsed treatment with JW576 (5 mm, 10 min).
Quantification of gel-resolved, labeled KIAA1363 (b) was performed
with ImageJ software and fluorescence intensity values were fit to
single-phase exponential decay models (a). c,d) KIAA1363 protein half-
life determination in DU145 cells as above in (a,b). Exponential decay
curves were generated in Prism 5 software. The data shown are
representative of two independent experiments.
Received: October 13, 2011
Published online: December 7, 2011
Keywords: ABPP · fluorescence imaging · fluorescent probes ·
.
KIAA1363 · serine hydrolases
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studies confirm that JW576 can be used to rapidly label
KIAA1363 activity with good selectivity in vivo.
In summary, we report herein a versatile fluorescent
probe for visualizing KIAA1363 activity in cancer cells and
in vivo. This agent JW576 is potent (sub-mm IC50 values in
cells) and selective for KIAA1363 and was used 1) in imaging
experiments to determine that KIAA1363 is largely, but not
exclusively localized to the ER in cancer cells, and 2) in pulse-
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KIAA1363 in cancer cells. That JW576 is also capable of
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