Identification of Tissue-Restricted Bioreaction Suitable for in Vivo Targeting by Fluorescent Substrate Library-Based Enzyme Discovery

Article abstract of DOI:10.1021/jacs.5b05884

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positi

Full text of DOI:10.1021/jacs.5b05884

Communication
pubs.acs.org/JACS
Identification of Tissue-Restricted Bioreaction Suitable for in Vivo
Targeting by Fluorescent Substrate Library-Based Enzyme Discovery
Kentaro Yoshioka,† Toru Komatsu,*,†,∥ Akihiro Nakada,† Jun Onagi,† Yugo Kuriki,†
Mitsuyasu Kawaguchi,†,# Takuya Terai,† Tasuku Ueno,† Kenjiro Hanaoka,† Tetsuo Nagano,§
and Yasuteru Urano*,†,‡,⊥
†Graduate School of Pharmaceutical Sciences, ‡Graduate School of Medicine, and §Open Innovation Center for Drug Discovery, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
∥PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
⊥CREST, Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo 100-0004, Japan
S
* Supporting Information
substrate probes, which enables direct readout of the enzymatic
activity with high sensitivity and high throughput,5 and we
ABSTRACT: Tissue-restricted bioreactions can be uti-
lized to design chemical-biological tools and prodrugs. We
have developed a fluorescent-substrate-library-based en-
zyme discovery approach to screen tissue extracts for
enzymatic activities of interest. Assay-positive candidate
proteins were identified by diced electrophoresis gel assay
followed by peptide mass fingerprinting. We discovered
that pyruvyl anilide is specifically hydrolyzed by carbox-
ylesterase 2 (CES2), which is predominantly localized in
the liver and kidney. We show that the pyruvyl targeting
group/CES2 enzyme pair can be used to deliver the 7-
amino-4-methylcoumarin fluorophore specifically to the
liver and kidney in vivo. Our screening approach should be
useful to find other masking group/enzyme pairs suitable
for development of fluorescent substrates and prodrugs.
searched for suitable activities by monitoring the susceptibility
of the probes to enzymatic reactions in extracts of tissue
samples. To our knowledge, such a strategy has never
previously been tried for the present purpose. Characterization
of enzymes mediating detected activities is potentially a
bottleneck, but our laboratory has recently developed an
efficient method, diced electrophoresis gel assay6 followed by
peptide mass fingerprinting, to characterize the target enzyme
of a given fluorescent substrate in biological samples. Here this
approach enabled us to discover that pyruvyl anilide is cleaved
in the liver and kidney by carboxylesterase 2 (CES2) with high
efficiency and high selectivity. To demonstrate its usefulness,
we show that the pyruvyl moiety/CES2 pair can be used to
deliver the 7-amino-4-methylcoumarin (AMC) fluorophore
specifically to the targeted tissues.
Fluorescent probe libraries for aminopeptidases,7 proteases,8
and oxidoreductases9 have been well-developed on the basis of
existing biochemical knowledge, but in the present work we
decided to focus on activities for hydrolysis of amide bonds
involving biologically occurring carboxylic acids other than
amino acids because they are applicable for the design of
functional small molecules and prodrugs and because enzymes
mediating these reactions have not been systematically studied
and characterized to date. We prepared a library of amides
based on the AMC fluorophore.10 Protection of the 7-amino
group quenches the fluorescence, and hydrolysis releases
fluorescent AMC. Representative structures and chemical
characteristics are shown in Scheme S1, Table S1, and Figure
1. Acetyl (1), propionyl (2), n-hexyl- (3), acryl- (4), benzoyl-
(5), 2-nitrophenacyl- (6), 4-nitrobenzoyl- (8), diglyoxylyl- (9),
and trifluoroacetyl-AMC (14) were prepared as derivatives of
general small-molecule carboxylic acids. Methyloxalyl-AMC
(13) was prepared as a representative of other nucleophiles.
Formyl- (7), pyruvyl- (10), and nicotinyl-AMC (12) were
prepared as controls for hydrolysis of biologically occurring
formyl and pyruvyl amides and nicotinamides. The N,N-
ne of the aims of chemical biology is to develop chemical
Otools that enable researchers to visualize or modulate
cellular functions in a spatiotemporally controlled manner by
utilizing specific bioreactions.1 For example, a bioreaction
commonly used for tool development is cleavage of the β-
galactopyranosyl group by β-galactosidase.2 However, in view
of the enormous number of potentially targetable enzymes in
the proteome,1c,3 the currently available choice of targeting
moiety/target enzyme pairs is extremely limited. In particular,
identification of selective bioreactions catalyzed by endogenous
enzymes with restricted tissue- or cell-type distribution is
urgently required to provide a basis for tissue- or cell-type-
specific delivery of imaging agents or functional molecules in
the human body without the need for gene manipulation.
Therefore, the aim of the work described here was to discover
bioreactions (targeting moiety/target enzyme pairs) with
potential applicability for the development of functional
molecules. The search for suitable reactions, especially efficient
ones, is not straightforward; it is not enough simply to compare
protein expression levels in different tissues, since enzymatic
activity is not necessarily directly related to the protein
expression level,1c,4 and the contribution of a particular enzyme
to the metabolism of individual substrates cannot be easily
estimated. Therefore, we employed a library of fluorescent
Received: June 7, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b05884
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 1. Fluorescence increase rates of fluorescent probes (10 μM)
after incubation with mouse tissue lysates (1 mg/mL) in phosphate
buffer (pH 7.4) containing CaCl2 and MgCl2.
diethylaminoacetyl group (11) was derived from the protective
group of the bioactive drug lidocaine.
Fluorometric assays of these probes were performed in
extracts of various mouse tissue lysates. Interestingly, we found
that the pyruvyl amide-type probe (10, Pyr-AMC) was
hydrolyzed with high specificity in liver and kidney lysates
(Figure 1). There are several reports on protease probes with
high tissue selectivity,1c but tissue-selective activity was rarely
seen among the fluorescent substrates for aminopeptidases that
we tested, such as Leu-AMC (for leucine aminopeptidase),
Met-AMC (for methionine aminopeptidase), and Ala-AMC
(for multiple targets) (Figure S1). Therefore, we found the case
of Pyr-AMC interesting. It is known that N-pyruvylated
peptides are generated by radical-mediated degradation of
proteins (oxidative stress),11 but to date no enzymes have been
reported to exhibit metabolic activity toward pyruvyl amides.
Thus, we used our recently developed diced electrophoresis gel
assay6 to identify the target enzyme of Pyr-AMC. After
optimizing the separation conditions of native electrophoresis,
we were able to detect two protein spots that exhibited
hydrolytic activity toward Pyr-AMC (Figures 2A,B and S2).
Each spot likely contains multiple proteins, and we used
peptide-mass-fingerprinting-based characterization to generate
a list of candidate target proteins, as summarized in Table S2.
Proteases such as cathepsin B and trypsin, which we expected
to have potential amide-cleaving activity, did not show
hydrolytic activity toward Pyr-AMC (Figure S3). Certain
carboxylesterases (CESs) appeared in both spots, and we
focused on this class of enzymes for further investigation since
some CESs are known to hydrolyze amides as well as esters.12
Indeed, the spots coincided with activity toward fluorescein
dibutyrate (FDBu), a general esterase probe6 (Figure 2C), and
the general CES inhibitor bis(4-nitrophenyl) phosphate
(BNPP) was confirmed to block the fluorescence increase of
Pyr-AMC (Figure S4). In order to further evaluate hydrolysis of
Pyr-AMC by CESs, we next prepared lysates of HeLa cells
transiently expressing various mouse CESs (Figure S5). Among
19 CES subtypes in mouse,13 we selected eight that are known
to be present in the liver (including members of all three major
subclasses, CES1, -2, and -3) and transfected their comple-
mentary DNAs into HeLa cells.
Figure 2. Characterization of CES2 as a pyruvyl anilide-hydrolyzing
enzyme. (A) CBB-staining image of a two-dimensional electrophoresis
gel of mouse liver lysate. (B, C) Results of two-dimensional diced
electrophoresis gel assay of mouse liver lysate with (B) Pyr-AMC (10
μM) or (C) FDBu (10 μM). Spots with white arrows in (C)
correspond to the spots detected in (B), while spots with white
arrowheads were not detected in (B). (D, E) Results of fluorometric
assays of (D) Pyr-AMC and (E) FDBu with HeLa cell lysate
expressing CES1, -2, and -3 isoforms (n = 3). * denotes significantly
increased activity compared with mock-transfected cells (P < 0.05).
Expression of the desired proteins was confirmed by the
observation of increased hydrolytic activity toward FDBu in the
lysates. We confirmed that fluorescence activation of Pyr-AMC
occurred only in lysates of CES2-expressing cells (Figure 2D).
Comparison of the activities with those in liver lysate by means
of one-dimensional and two-dimensional diced electrophoresis
gel assay (Figures S6 and S7) as well as the results of Western
blotting confirmed that the major target of Pyr-AMC is CES2.
It is noteworthy that whereas FDBu showed elevated activity
with all classes of esterases (Figure 2E), Pyr-AMC showed
elevated activity only in CES2-expressing lysates; in other
words, Pyr-AMC showed complete selectivity for CES2 among
various CESs. We studied the substrate scope of various pyruvyl
anilide-bearing molecules for CES2 and found that pyruvyl
anilides with fluorophores other than AMC were also good
substrates of CES2 with comparable selectivities (Figure S8),
whereas pyruvyl amides with aliphatic amines (such as naturally
occurring pyruvyl peptides) were not (Figure S9).
Therefore, the pyruvyl anilide structure seems to be
necessary and sufficient for targeting of CES2. Carboxyles-
terases are involved in metabolism of exogenous compounds14
and lipid metabolism,15 but their physiological roles are not
fully understood. However, CES2 levels are altered in various
B
DOI: 10.1021/jacs.5b05884
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
pathological states,16 and some tumor cells express elevated
levels of CES2.17 After confirming the selectivity of Pyr-AMC
for CES2 over CES1 in the case of human carboxylesterases
(Figure S10), we tested whether Pyr-AMC can be used to
detect CES2 activity at the cellular level. We prepared lysates of
seven cancer cell lines and studied the correlation between the
CES2 expression level and the fluorescence increase of Pyr-
AMC. We confirmed that marked fluorescence was generated
by lysates of HepG2 and SKOV3, cell lines that are reported to
express high levels of CES217 (Figure 3A). Although we cannot
no Pyr-AMC hydrolytic activity was detected in mouse serum
(Figure S11), this might be due to leakage of AMC from the
above two tissues.
Tissue-selective activation of functional molecules for
imaging purposes has already been achieved by targeting β-
galactosidase as a reporter enzyme,18 but here we have
succeeded in tissue-selective activation by utilizing an
endogenous, tissue-restricted enzyme without the need for
any genetic modification. These results are in sharp contrast to
the results of in vivo targeting of AMC with a leucyl group
(Leu-AMC), which proved unsuccessful; AMC was detected in
all of the tissues tested, and the highest concentration was
observed in plasma. This is probably due to the ubiquitous
presence of the targeted enzyme (presumably leucine amino-
peptidases). We also synthesized a pyruvyl-amidated methyl
ester derivative of p-aminosalicylic acid (PAS), an NF-κB
inhibitor that has also been used to treat tuberculosis, and
confirmed that the prodrug, PAS methyl ester, was released in
the presence of CES2 (Figure S12). This result suggests that
the discovered bioreaction would be available for delivery of
druglike molecules. Several commercial prodrugs are activated
by CESs (e.g., capecitabine17 and iriniotecan19), but they are
metabolized by both CES1 and CES2 in the human body. Our
reaction was highly selective for CES2 in both mouse and
human. We think that it led to the stability of compounds in
blood and nontargeted tissues where CES2 was not present
(Figure S11) and should enable successful utilization of this
reaction for tissue-selective delivery of drugs by intravascular
injection; a sufficiently high reaction rate might also be a
contributory factor (Figure S13). It should be noted that CESs
are also present in digestive tissues, such as the stomach and
small and large intestines,20 so the possibility that activities in
those tissues contribute to the metabolism of Pyr-AMC should
be considered, especially in the case of oral administration. In
any event, we believe that our screening system will also be
applicable for the discovery of other novel enzyme−substrate
pairs suitable for targeting other tissues. Studies along the line
are already in progress.
In conclusion, we have utilized a fluorescent-substrate-
library-based enzyme discovery approach to identify a novel
tissue-restricted bioreaction, i.e., cleavage of pyruvyl anilide by
CES2 in the liver and kidney. We showed that the pyruvyl
targeting group/CES2 enzyme pair can be used to deliver the
AMC fluorophore specifically to the liver and kidney in vivo.
We also confirmed that CES2 hydrolyzes pyruvyl-amidated
PAS methyl ester, indicating that other functional molecules
should be deliverable with this system. This work illustrates the
ability of the target discovery approach to identify novel
bioreactions suitable as a basis for the future development of a
range of chemical-biological tools, such as fluorescent substrates
and prodrugs. We believe that this approach will be broadly
useful to characterize novel protein functions in the enzymome
(the complete set of enzymes active in a cell)21 and also to
identify characteristic target candidates in various pathological
states.
Figure 3. CES2-mediated hydrolysis of pyruvyl anilides for in vivo
targeting of functional molecules. (A) Fluorescence intensities of Pyr-
AMC (1 μM) in lysates (0.1 mg/mL) of various cells. (B) Tissue
distribution of 7-amino-4-methylcoumarin (AMC) 10 min after
intravenous injection of AMC, Leu-AMC, or Pyr-AMC (5 mg/kg)
into mice (n = 3).
completely exclude a possible contribution of differential
expression of hydrolytic enzymes other than CES2 to the
fluorescence increases, these results support the idea that Pyr-
AMC can detect endogenous activity of CES2 with sufficient
sensitivity and selectivity for practical applications.
Finally, we examined the availability of this bioreaction for in
vivo delivery of functional molecules. After intravenous
injection of Pyr-AMC into mice, the formation of fluorescent
AMC was observed exclusively in the liver and kidney, where
CES2 expression levels are high (Figure 3B). A low
concentration of AMC was also detected in plasma, but since
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b05884.
Methods and supporting schemes, tables, and figures
(PDF)
C
DOI: 10.1021/jacs.5b05884
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(11) Cecarini, V.; Gee, J.; Fioretti, E.; Amici, M.; Angeletti, M.;
Eleuteri, A. M.; Keller, J. N. Biochim. Biophys. Acta, Mol. Cell Res. 2007,
1773, 93−104.
AUTHOR INFORMATION
■
Corresponding Authors
*tkomatsu@mol.f.u-tokyo.ac.jp
*uranokun@m.u-tokyo.ac.jp
(12) Alexson, S. E.; Diczfalusy, M.; Halldin, M.; Swedmark, S. Drug
Metab. Dispos. 2002, 30, 643−7.
(13) Quinney, S. K.; Sanghani, S. P.; Davis, W. I.; Hurley, T. D.; Sun,
Z.; Murry, D. J.; Bosron, W. F. J. Pharmacol. Exp. Ther. 2005, 313,
1011−6.
(14) Holmes, R. S.; Wright, M. W.; Laulederkind, S. J. F.; Cox, L. A.;
Hosokawa, M.; Imai, T.; Ishibashi, S.; Lehner, R.; Miyazaki, M.;
Perkins, E. J.; Potter, P. M.; Redinbo, M. R.; Robert, J.; Satoh, T.;
Yamashita, T.; Yan, B.; Yokoi, T.; Zechner, R.; Maltais, L. J. Mamm.
Genome 2010, 21, 427−41.
Present Address
#M.K.: Graduate School of Pharmaceutical Sciences, Nagoya
City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-
8603, Japan.
Notes
The authors declare no competing financial interest.
(15) Dominguez, E.; Galmozzi, A.; Chang, J. W.; Hsu, K. L.; Pawlak,
J.; Li, W.; Godio, C.; Thomas, J.; Partida, D.; Niessen, S.; O’Brien, P.
E.; Russell, A. P.; Watt, M. J.; Nomura, D. K.; Cravatt, B. F.; Saez, E.
Nat. Chem. Biol. 2014, 10, 113−21.
ACKNOWLEDGMENTS
■
We thank Dr. Masahiro Shimoda and Ms. Makiko Kawamura
for preparing the plates to perform diced electrophoresis gel
assays. LC−MS/MS data were collected by APRO Life Science
Institute under contract. This work was supported in part by
MEXT (22000006 to T.N., 24689003 and 24659042 to K.H.,
and 24655147, 15H05371, and 15K14937 to T.K.), JST (K.H.
and T.K.), and AMED (Y.U.). T.K. was also supported by the
Mochida Memorial Foundation for Medical and Pharmaceutical
Research.
(16) (a) Tang, X.; Wu, H.; Wu, Z.; Wang, G.; Wang, Z.; Zhu, D.
Cancer Invest. 2008, 26, 178−81. (b) Cai, L.; Tang, X.; Guo, L.; An, Y.;
Wang, Y.; Zheng, J. Tumori 2009, 95, 473−8.
(17) Pratt, S. E.; Durland-Busbice, S.; Shepard, R. L.; Heinz-Taheny,
K.; Iversen, P. W.; Dantzig, A. H. Clin. Cancer Res. 2013, 19, 1159−68.
(18) Oushiki, D.; Kojima, H.; Takahashi, Y.; Komatsu, T.; Terai, T.;
Hanaoka, K.; Nishikawa, M.; Takakura, Y.; Nagano, T. Anal. Chem.
2012, 84, 4404−10.
(19) Humerickhouse, R.; Lohrbach, K.; Li, L.; Bosron, W. F.; Dolan,
M. E. Cancer Res. 2000, 60, 1189−92.
(20) (a) Bachovchin, D. A.; Ji, T.; Li, W.; Simon, G. M.; Blankman, J.
L.; Adibekian, A.; Hoover, H.; Niessen, S.; Cravatt, B. F. Proc. Natl.
Acad. Sci. U. S. A. 2010, 107, 20941−6. (b) Xie, M.; Yang, D.; Liu, L.;
Xue, B.; Yan, B. Drug Metab. Dispos. 2002, 30, 541−7.
(21) Kahl, G. The Dictionary of Genomics, Transcriptomics and
Proteomics; Wiley-VCH: Weinheim, Germany, 2015.
REFERENCES
■
(1) (a) Johnsson, N.; Johnsson, K. ACS Chem. Biol. 2007, 2, 31−8.
(b) Johnsson, K. Nat. Chem. Biol. 2009, 5, 63−5. (c) Edgington, L. E.;
Verdoes, M.; Bogyo, M. Curr. Opin. Chem. Biol. 2011, 15, 798−805.
(2) (a) Kamiya, M.; Kobayashi, H.; Hama, Y.; Koyama, Y.; Bernardo,
M.; Nagano, T.; Choyke, P. L.; Urano, Y. J. Am. Chem. Soc. 2007, 129,
3918−29. (b) Jain, V. K.; Magrath, I. T. Anal. Biochem. 1991, 199,
119−24. (c) Koide, Y.; Urano, Y.; Yatsushige, A.; Hanaoka, K.; Terai,
T.; Nagano, T. J. Am. Chem. Soc. 2009, 131, 6058−9. (d) Hanaoka, K.;
Kikuchi, K.; Terai, T.; Komatsu, T.; Nagano, T. Chem. - Eur. J. 2008,
14, 987−95.
(3) (a) Webb, E. C. Enzyme Nomenclature 1992: Recommendations of
the Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology on the Nomenclature and Classification of
Enzymes; International Union of Biochemistry and Molecular Biology,
1992. (b) Long, J. Z.; Cravatt, B. F. Chem. Rev. 2011, 111, 6022−63.
(c) Komatsu, T.; Urano, Y. Anal. Sci. 2015, 31, 257−65.
(4) Saghatelian, A.; Cravatt, B. F. Nat. Chem. Biol. 2005, 1, 130−42.
(5) (a) Er, J. C.; Leong, C.; Teoh, C. L.; Yuan, Q.; Merchant, P.;
Dunn, M.; Sulzer, D.; Sames, D.; Bhinge, A.; Kim, D.; Kim, S. M.;
Yoon, M. H.; Stanton, L. W.; Je, S. H.; Yun, S. W.; Chang, Y. T. Angew.
Chem., Int. Ed. 2015, 54, 2442−6. (b) Wagner, B. K.; Carrinski, H. A.;
Ahn, Y. H.; Kim, Y. K.; Gilbert, T. J.; Fomina, D. A.; Schreiber, S. L.;
Chang, Y. T.; Clemons, P. A. J. Am. Chem. Soc. 2008, 130, 4208−9.
(6) Komatsu, T.; Hanaoka, K.; Adibekian, A.; Yoshioka, K.; Terai, T.;
Ueno, T.; Kawaguchi, M.; Cravatt, B. F.; Nagano, T. J. Am. Chem. Soc.
2013, 135, 6002−5.
(7) (a) Backes, B. J.; Harris, J. L.; Leonetti, F.; Craik, C. S.; Ellman, J.
A. Nat. Biotechnol. 2000, 18, 187−93. (b) Drag, M.; Bogyo, M.;
Ellman, J. A.; Salvesen, G. S. J. Biol. Chem. 2010, 285, 3310−8.
(8) (a) Kato, D.; Boatright, K. M.; Berger, A. B.; Nazif, T.; Blum, G.;
Ryan, C.; Chehade, K. A. H.; Salvesen, G. S.; Bogyo, M. Nat. Chem.
Biol. 2005, 1, 33−8. (b) Thomas, D. A.; Francis, P.; Smith, C.;
Ratcliffe, S.; Ede, N. J.; Kay, C.; Wayne, G.; Martin, S. L.; Moore, K.;
Amour, A.; Hooper, N. M. Proteomics 2006, 6, 2112−20.
(9) (a) Yee, D. J.; Balsanek, V.; Bauman, D. R.; Penning, T. M.;
Sames, D. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 13304−9.
(b) Froemming, M. K.; Sames, D. J. Am. Chem. Soc. 2007, 129,
14518−22.
(10) (a) Zimmerman, M.; Yurewicz, E.; Patel, G. Anal. Biochem.
1976, 70, 258−62. (b) Zimmerman, M.; Ashe, B.; Yurewicz, E. C.;
Patel, G. Anal. Biochem. 1977, 78, 47−51.
D
DOI: 10.1021/jacs.5b05884
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX