Small-molecule probe using dual signals to monitor leucine aminopeptidase activity

Article abstract of DOI:10.1016/j.bmcl.2011.02.068

Leucine aminopeptidases (LAPs) are widely distributed in organisms from bacteria to humans, and play crucial roles in cell maintenance and cell growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this Letter, we

Full text of DOI:10.1016/j.bmcl.2011.02.068

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2403–2405
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Bioorganic & Medicinal Chemistry Letters
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Small-molecule probe using dual signals to monitor leucine
aminopeptidase activity
Hey Young Yoon a, So Hee Shim b, Luck Ju Baek b, Jong-In Hong a,
⇑
a Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
b Department of Microbiology and Institute for Viral Diseases, College of Medicine, Korea University, Seoul 136-705, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Leucine aminopeptidases (LAPs) are widely distributed in organisms from bacteria to humans, and play
crucial roles in cell maintenance and cell growth. Thus, assays for LAP are necessary for measuring its
activity and inhibitor potency. In this Letter, we report a small-molecule probe which exhibits colorimet-
ric and fluorogenic changes according to LAP activity.
Received 24 December 2010
Revised 15 February 2011
Accepted 16 February 2011
Available online 18 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Leucine aminopeptidases
Probe
Dual signals
2-Dicyanomethylene-3-cyano-2,5-
dihydrofuran (DCDHF) unit
Aminopeptidases play important roles as transcriptional repres-
sors, site-specific recombination factors, and viral or toxin recep-
tors, as well as being proteolytic enzymes for the N-terminus of
proteins and bioactive peptides in bacteria and animals. Leucine
aminopeptidases (LAPs) are one of the exopeptidases which cata-
lyze the hydrolysis of the N-terminal leucine residues of proteins
or peptides. Some LAPs are key enzymes and are able to affect di-
verse biological and physiological phenomena.1–4 They could
therefore be used as diagnostic or prognostic biomarkers, and as-
says for their activity are necessary. Several assays for LAP activi-
ties have been developed.4–9 Known probes for monitoring LAP
activities consist of a fluorescent reporter and an amide group cou-
pled with leucine, and they can exhibit colorimetric and fluoro-
genic signals on interaction with LAPs.4 However, they have
short excitation and emission wavelengths, making it hard to mon-
itor LAP activity in living cells, and they need large amounts of LAP
for high-throughput screening for potent inhibitors. To address
these problems, we developed a probe, using a 2-dicyanomethyl-
emission by reducing the donor ability of the NH moiety in
DCDHF–Leu. However, DCDHF–Leu is expected to show color
changes and fluorescence turn-on when the leucine is uncapped
by treatment with LAP, forming DCDHF–NH2 (Scheme 1).
We evaluated the optical response to LAP activity using
DCDHF–Leu in vitro. When it was excited at 525 nm, DCDHF–Leu
was non-fluorescent, but DCDHF–NH2 showed significant fluores-
cence enhancement in HEPES buffer at pH 7.4 ( Fig. 1A). The
emission spectrum of DCDHF–Leu after treatment with porcine
kidney microsomal LAP (PKLAP) was examined with time
(Fig. 1B). The fluorescence intensity at 605 nm increased by up to
10-fold, and was saturated in 30 min. This indicates that PKLAP
removes the leucine residue from DCDHF–Leu to form
ene-3-cyano-2,5-dihydrofuran (DCDHF) unit as
a p-electron-
accepting unit, for fluorescent measurement of LAP activity and
inhibitor potency at longer wavelengths.
We designed
leucine-linked
a probe which has a DCDHF unit and a
p
-conjugated system (DCDHF–Leu). This probe has
good cell permeability and can be applied in bioimaging.10–13 The
capped amine functionality of leucine could block fluorescence
⇑
Corresponding author.
E-mail address: jihong@snu.ac.kr (J.-I. Hong).
Scheme 1. Probe based on the 2-dicyanomethylene-3-cyano-2,5-dihydrofuran
(DCDHF) unit.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.068

2404
H. Young Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2403–2405
Figure 1. (A) Fluorescence spectra of DCDHF–NH2 and DCDHF–Leu; (B) time-dependent fluorescence changes of a DCDHF–Leu probe (10
l
M) with PKLAP (0.2
lg/mL) at
605 nm; (C) color change of a DCDHF–Leu probe after 30 min incubation with PKLAP: 1, 20
PKLAP.
lM DCDHF–NH2; 2, 20
lM DCDHF–Leu; 3, 20
lM DCDHF–Leu with 0.2 l
g/mL
Table 1
Enzyme kinetics data
Substrate
Vmax
(
l
mol/min mg)
Km
3000 200
65
8.87 1.72
(
l
M)
IC50 of bestatinb
(lM)
l-Leucine-p-nitroanilidea
3.9 0.2
7.7 0.2
6.64 0.46
25
7
4
1
N-(6-Methoxypyridine-3-yl), (S)-2-amino-4-methylpentanamidea
4
DCDHF–Leu
4.41 0.61
a
Ref. 4.
b
Measured values using each substrate.
DCDHF–NH2 within minutes, which means that DCDHF–Leu is a
good fluorogenic substrate for PKLAP. Moreover, incubation of
DCDHF–Leu with LAP for 30 min triggered a solution color change
from yellow to red. It was demonstrated that PKLAP activity using
the probe could also be observed by the naked eye ( Fig. 1C).
DCDHF–Leu is therefore an efficient probe that can measure LAP
activity by fluorescent and color changes.
To determine the enzyme kinetics, the fluorescence intensity
was examined with different concentrations of DCDHF–Leu and a
constant amount of PKLAP. Concentrations of DCDHF–Leu from
determined to be 6.64 0.46
stant (Km) was 8.87 1.72
l
mol/min mg and the Michaelis con-
l
M. Compared to previously reported
data (Table 1),4 DCDHF–Leu has a similar Vmax and smaller Km.
These data indicate that DCDHF–Leu could bind to LAP more
tightly, and that it is a better substrate for LAP than other reported
or commercial substrates.
To explore whether the DCDHF–Leu probe can measure inhibi-
tory potency, we determined the inhibition potency (IC50) of best-
atin, which is known to be a slow-binding competitive inhibitor of
LAP.14–16 A constant amount of PKLAP (0.2
l
g/mL) was incubated
with various concentrations of bestatin, from 0.6 to 50 M, for
10 min, prior to addition of 10 M DCDHF–Leu. The maximum
1.2 to 50
l
M, and 0.2
lg/mL PKLAP in 10 mM HEPES buffer at pH
l
7.4, were used (Fig. S3). The maximum velocity (Vmax) was
l
Figure 2. (A) fluorescence decrease of DCDHF–Leu with bestatin concentration (0.6, 1.2, 2.5, 5, 10, 20 and 50 lM); (B). Inhibitory potency.

H. Young Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2403–2405
2405
temperature. The HCT 116 cells without bestatin showed red fluo-
rescent imaging, indicating the presence of DCDHF–NH2 from reac-
tion with LAP; the other set, with bestatin, which could block the
LAP activity, was non-fluorescent (Fig. 3).The fluorescent intensity
of HCT 116 cell lysates incubated with 20 lM DCDHF–Leu and no
bestatin was about six-fold higher than that of HCT 116 cell lysates
incubated with the probe and bestatin (Fig. S4). The same results
were obtained from the cell lysate and the living cell, indicating
that DCDHF–Leu has good cell permeability. All our experiments
reveal that DCDHF–Leu is an efficient probe for monitoring LAP
activity in living cells.
In conclusion, we have developed a fluorescent probe (DCDHF–
Leu) that can monitor LAP activity in living cells. It was demon-
strated that DCDHF–Leu showed significant fluorescent enhance-
ment and a color change on reaction with LAP. In addition, it was
illustrated that DCDHF–Leu was an efficient probe for measuring
the inhibitory potency of LAP inhibitors.
Acknowledgement
This study was supported by the NRF Grants funded by the
MEST (No. 2009-0080734, 2010-0001842 for the Center for Next
Generation Dye-sensitized Solar Cells).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.068.
References and notes
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fluorescence intensity at 605 nm and the initial DCDHF–Leu veloc-
ity with PKLAP decreased dose-dependently (Fig. 2). The IC50 ob-
tained in this experiment is 4.41 0.61 lM, which is the same
order of magnitude as previously reported data (Table 1).4 Our
experiment reveals that DCDHF–Leu is an excellent probe, which
can detect compounds with high inhibition potency for LAP, and
also enables high-throughput screening.
Finally, to demonstrate the potential practical applications of
DCDHF–Leu, we applied the probe to a living cell for fluorescence
imaging. DCDHF–NH2 is a red fluorescent substrate, which has
longer excitation and emission wavelengths than previously
known probes.4 Two sets of HCT 116 cells were incubated with
16. Stamper, C. C.; Bienvenue, D. L.; Bennett, B.; Ringe, D.; Petsko, G. A.; Holz, R. C.
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20 lM DCDHF–Leu, with and without bestatin, for 20 min at room