Multicolor imaging of endoplasmic reticulum-located esterase as a prodrug activation enzyme

Article abstract of DOI:10.1021/ml400398t

The carboxylesterase families of enzymes are key participants in phase I drug metabolism processes. Carboxylesterase families 1 and 2 are of particular clinical relevance. These enzymes produce endoplasmic reticulum localization signals, are primarily localized in the endoplasmic reticulum, and hydrolyze a wide range of ester-containing prodrugs into an activated form. In order to detect enzymes belonging to both families, we developed an optical multicolor imaging technique, which provides a distinct color window for multicolor imaging. This technique required the design and synthesis of three new mechanistic colored probes that fluoresce red, green, or blue and are based on the quinone methide cleavage process. These activity-based probes allow rapid and clear visualization with high specificity against the endoplasmic reticulum in cultured cells based on endoplasmic reticulum localized esterases including both families of carboxylesterase enzymes.

Full text of DOI:10.1021/ml400398t

Letter
pubs.acs.org/acsmedchemlett
Multicolor Imaging of Endoplasmic Reticulum-Located Esterase As a
Prodrug Activation Enzyme
Wataru Hakamata,* Saori Tamura, Takako Hirano, and Toshiyuki Nishio
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi,
Kanagawa 252-0880, Japan
S
* Supporting Information
ABSTRACT: The carboxylesterase families of enzymes are key
participants in phase I drug metabolism processes. Carboxylesterase
families 1 and 2 are of particular clinical relevance. These enzymes
produce endoplasmic reticulum localization signals, are primarily
localized in the endoplasmic reticulum, and hydrolyze a wide range of
ester-containing prodrugs into an activated form. In order to detect
enzymes belonging to both families, we developed an optical multicolor
imaging technique, which provides a distinct color window for multicolor
imaging. This technique required the design and synthesis of three new
mechanistic colored probes that fluoresce red, green, or blue and are
based on the quinone methide cleavage process. These activity-based
probes allow rapid and clear visualization with high specificity against the endoplasmic reticulum in cultured cells based on
endoplasmic reticulum localized esterases including both families of carboxylesterase enzymes.
KEYWORDS: Prodrug, carboxylesterase, fluorescent probe, quinone methide cleavage
arboxylesterases (CEs) metabolize numerous exogenous
reported an ER-localized CE-specific fluorescence probe, 1, for
Cand endogenous ester-containing compounds and play a detecting CE activity using the quinone methide cleavage
process, which is a 1,6-elimination reaction, as shown in Figure
1A. The fluorogenic chemical transformation of 1 triggered by
CEs in the ER occurs through two reactions, ester hydrolysis
and quinone methide cleavage, which are spontaneous and
irreversible in cells. After hydrolysis, released resorufin (EX, 571
nm; EM, 585 nm) from 1 specifically stained ERs with bright
red fluorescence in a human fibrosarcoma cell line, HT-1080,
and kidney cells of the African green monkey, COS-1.9
Previous research on the location of CEs in human cells
showed that CEs are located in the ER10 and cytoplasm.11−13
Interestingly, probe 1 is an ER-specific dye, whereas
commercially available ester-protected fluorescein derivatives
activated by CEs also dye the cytoplasm.
Multicolor imaging is used for the in vitro microscopic
imaging of specific enzyme activity. These in vitro microscope
studies are generally performed using combinations of
commercially available red, green. and blue fluorescent probes.
The images are taken separately, one color at a time, using an
appropriate filter set, and then the colors are merged during
postprocessing to construct a multicolor image. Multicolor
imaging of CES activity in the ER at the cellular level is clearly
of interest and could be accomplished based on our previous
research.9 To develop probes for multicolor imaging of CE
activity in the ER, TFMU (EX, 385 nm; EM, 502 nm) and
role in a broad range of biological processes. CEs are a key
participant in phase I drug metabolism processes by catalyzing
the hydrolysis of various prodrugs. This is a well-established
strategy for improving the physicochemical, biopharmaceutical,
or pharmacokinetic properties of pharmacologically potent
compounds.1,2 Another interesting bioactive phenomenon,
recently reported by Pezacki et al., is that carboxylesterase 1
is a host factor involved in the hepatitis C virus life cycle.3 The
CEs are classified into five families (CES1, CES2, CES3, CES4,
and CES5) according to the homology of their amino acid
sequence.4 The hydrolytic abilities of the CES1 and CES2
families are important for the bioconversion of ester-containing
prodrugs. Both families contain the HXEL (His-X-Glu-Leu)
sequence at the C-terminal, which can bind with the KDEL
(Lys-Asp-Glu-Leu) sequence in the endoplasmic reticulum
(ER) protein retention receptor. This binding reaction is
essential for retention of the protein on the luminal site of the
ER. Ester-containing prodrugs that pass through the cell
membrane and ER membrane by simple diffusion are therefore
hydrolyzed to their activated forms by CES1 and/or CES2
family enzymes in the ER. The activation of prodrugs by these
enzymes provides a possible means for overcoming various
barriers in drug formulation and delivery such as poor aqueous
solubility, chemical instability, insufficient oral absorption, rapid
presystemic metabolism, inadequate brain penetration, toxicity,
and local irritation.5−8 However, despite their importance in
drug activation, biological activity imaging of CEs in the ER at
the cellular level has yet to be fully established. Our group has
Received: October 5, 2013
Accepted: January 16, 2014
Published: January 16, 2014
© 2014 American Chemical Society
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Figure 1. Schematic illustration of ER-specific imaging using the strong fluorescence provided by the probes following quinone methide cleavage
(A). Structure and fluorescence properties of probes 1−4 (B).
Figure 2. Synthetic scheme for probes 2−4.
2MeTG (EX, 491 nm; EM, 510 nm)14−16 were chosen as the
blue and green fluorophores, respectively. The fluorophores
resorufin, TFMU and 2MeTG each have distinct excitation and
emission wavelengths, excellent fluorescence quantum yields,
and low pH-dependent change in fluorescence, making them
suitable for multicolor imaging at the cellular level.
provide multicolor imaging of CES1 and CES2 activity as
prodrug activators in the ER. The substrate specificities of
CES1 and CES2 are significantly different: CES1 has a broad
substrate specificity, whereas CES2 does not accept bulky acyl
groups as substrate.17 All the probes contain a large alcohol
group, making these probes likely CES2 substrates.
Here we describe the design, synthesis, and photochemical
and biological properties of fluorescence probes 1−4. The
probes were used to image CEs in three human cell lines, as
shown in Figure 1B. The results show that this approach can
Probes 1−4 are compatible for use as fluorescent dyes in cells
as they exhibit appropriate membrane permeability. Waring18
reported that, to achieve good membrane permeation, a logD
value of >1.7 is required for compounds with a molecular
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4.03, 8.10 and 7.95, respectively, between pH 7 to 8, have good
membrane permeability.19 The synthesis of probes 2−4 is
presented in Figure 2. The fluorescence spectra of probes 1−4
are provided in the Supporting Information.
To be useful as CES activity multicolor imaging tools in cells,
the emission of probes 1−4 should not overlap the excitation
conditions of the parent fluorophores resorufin, TFMU and
2MeTG. To observe the emission spectra of each fluorophore
and probes 1−4 in PBS (−), the emission spectra were
measured. Probes 1−4 showed almost no emission. For
synthesis details, refer to Supporting Information.
We investigated the release of each fluorophore (TFMU and
2MeTG) from probes 2−4 activated by porcine liver esterase in
vitro. The release of the fluorescence signal of each fluorophore
was clearly observed when probes 2−4 were coincubated with
the esterase. Plots of fluorescence intensity vs incubation time
(see Supporting Information) show pseudo-first order kinetics
for all the probes. The results indicate that the esterase shows
appropriate specific activity (5.91, 3.51, and 4.03 units/mg
protein) against probes 2−4, respectively. These results and
comparable results using probe 19 show that the enzyme
catalyzes probes 1−4 to release each fluorophore via a two-step
reaction: ester hydrolysis and spontaneous quinone methide
cleavage. These two reactions are not dependent on the
fluorophore structure under the conditions used. Therefore,
differences in the structure of the fluorophore do not affect the
quinone methide reaction in aqueous media. For enzyme
kinetics details, refer to Supporting Information.
Figure 3. Confocal laser scanning microscopy fluorescence images of
three cell lines stained with probes 1−4.
weight of 350−400, a logD value of >3.1 is required for
compounds with a molecular weight of 400−450, and a logD
value of >3.4 is required for compounds with a molecular
weight of 450−500. This has been demonstrated using Caco-2
cell data from AstraZeneca. Probes 1−4, which have molecular
weights of 389, 406, 479, and 479 and ClogD values of 4.87,
Finally, to demonstrate the utility of probes 1−4 for
multicolor imaging of CEs in ERs in three human cell lines,
Figure 4. Fluorescence images of probe 1, ER-Tracker, merged fluorescence image, and differential interference contrast microscopy (DIC) images
of three kinds of cell lines.
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we characterized the dynamics between localization and
enzyme activity. We introduced probes 1−4 into the cells
across the cell membrane and the ER membranes. The imaging
potential of 1−4 based on CE activity was evaluated in cell
culture of a human fibrosarcoma cell line, HT-1080, a human
neuroblastoma cell line, SK-N-SH, and a human epithelial
carcinoma cell line, HeLa. Each cell line was incubated with 5
μM probe 1, 2, 3 or 4 for 30 min in the presence of Hoechst
33258 as a nuclear fluorescence dye. Note that probe 2 is
excited at the same wavelength as Hoechst 33258. Differential
interference contrast microscope images and fluorescence
images were taken using confocal laser scanning microscopy.
The fluorescence signal of probes 1−4 indicated the intra-
cellular accumulation of resorufin, TFMU, and 2MeTG around
the nucleus, which was dyed with Hoechst 33258; typical
images showing the reticular structure of the ER are shown in
Figure 3. These images indicated that probes 1−4 had high
selectivity for the ERs of all the cell lines tested and provided
bright fluorescence. These results show that the cell enzyme
transformations and imaging of ERs by probes 1−4 do not
depend on the fluorophore structures and the ester structures.
We thus succeeded in optically separating the fluorescence
signal from probes 1−4, allowing us to visualize esterase activity
in the ER. On the basis of these results, many other
fluorophores with different structures may prove useful in
imaging studies and provide the possibility of additional
fluorescence colors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures and characterization of
compounds, in vitro enzyme assays, and cell culture assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(W.H.) E-mail: hakamata.wataru@nihon-u.ac.jp.
Funding
This research was partly supported by the Ministry of
Education, Science, Sports and Culture, by a Grant-in-Aid for
Young Scientists (B) (No. 17790097) to W.H., and Health and
Labour Sciences Research Grants for Research on HIV/AIDS
to W.H. from the Ministry of Health, Labour and Welfare,
Japan.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
CE, carboxylesterase; ER, endoplasmic reticulum; EX,
excitation wavelength; EM, emission wavelength; TFMU, 4-
trifluoromethylumbelliferone; 2MeTG, 2-methyl TokyoGreen;
PBS, phosphate buffered saline
Next, to conclusively determine the staining areas of these
activity-based probes, the three human cell lines were stained
simultaneously with probe 1 and a commercially available ER-
specific fluorescence dye, ER-Tracker Blue-White DPX dye
(EX, 374 nm; EM, 430 nm). Probe 1 was used as a typical
resorufin red fluorophore probe that possesses different
fluorescence excitation and emission wavelengths from ER-
Tracker. Figure 4 summarizes the results of the staining areas of
both dyes in the three cell lines. The staining area obtained
using probe 1 is identical to that obtained using ER-Tracker at
the same time. Imaging with probe 1 substantiated the
conclusion that these probes stain the ER with high specificity.
Clearly, probes 1−4 hold promise as ER-specific fluorescent
probes for esterase activity containing CEs. However, the
amount of fluorescence from these probes is highly dependent
on the expression level of the esterases. The probes should be
required to have high specificity for the CEs. Finally, we used
multicolor imaging of highly expressed esterase activity located
in the ERs using probes 1−4 to hydrolyze various prodrugs.
The methods can be used in the ERs of different human cell
types to image esterase activity containing CEs.
CE enzymes are one of the major determinants of the
metabolism and disposition of numerous prodrugs hydrolyzed
and activated in the ER. Thus, elucidating the mechanism
governing CE enzyme activity using small molecules will
significantly impact rational drug design and the future
development of prodrugs. These activity-based probes could
provide a basis for the design and development of the
physicochemical, biopharmaceutical, or pharmacokinetic prop-
erties of pharmacologically potent compounds as novel
therapeutics when activated by CEs. We envision the
application of these activity-based probes in the quantitation
of CE activity, the functional analysis of CEs, and the screening
of CE inhibitors at the cellular level.
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