Fluorescent bioprobes for visualization of puromycin-sensitive aminopeptidase in living cells.

Article abstract of DOI:10.1016/S0960-894X(02)00845-4

Non-peptide, small-molecular, non-competitive inhibitors of puromycin-sensitive aminopeptidase (PSA), that is, 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 3) and its 1N-methyl analogue (MPAQ-22 4), were structurally modified to afford fluor

Full text of DOI:10.1016/S0960-894X(02)00845-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 83–86
Fluorescent Bioprobes for Visualization of Puromycin-Sensitive
Aminopeptidase in Living Cells
Hiroki Kakuta, Yukiko Koiso, Kazuo Nagasawa and Yuichi Hashimoto*
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Received 30 August 2002; accepted 1October 2002
Abstract—Non-peptide, small-molecular, non-competitive inhibitors of puromycin-sensitive aminopeptidase (PSA), that is, 3-(2,6-
diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 3) and its 1N-methyl analogue (MPAQ-22 4), were structurally modified to
afford fluorescent bioprobes, ANTAQ (5) and DAMPAQ-22 (6). The cellular localization of PSA could be visualized by the use of
these fluorescent bioprobes.
# 2002 Elsevier Science Ltd. All rights reserved.
Puromycin-sensitive
aminopeptidase
(PSA:
EC
For the investigation of the molecular/cell-level
mechanism(s) of these PSA inhibitors and the physio-
logical role(s) of PSA, preparation of PSA-specific
bioprobes which are applicable to living cells is
required. Here, we report the preparation of fluorescent
bioprobes specific to PSA and their application to the
visualization of PSA in living cells.
3.4.11.14), which is a neutral aminopeptidase with a
substrate specificity similar to that of aminopeptidase N
(APN), is distributed mainly in the brain and neu-
rons.1À7 Though this enzyme was first isolated as a can-
didate for enkephalinase,1À3,5 and it has been reported
that PSA gene-deficient mice obtained by a mouse gene-
trapping method show increased anxiety and impaired
pain response,8 the physiological role(s)/function(s) of
PSA remain unclear because of the low substrate speci-
ficity of the enzyme and the lack of specific inhibitors.5À8
The localization of PSA at the cell structural level is still
not known.
We chose PAQ-22 (3) and MPAQ-22 (4) as pharmaco-
phores because of their superior chemical stability and
bioavailability compared to those of PIQ-22 (2).12,16
Recently, we have reported potent non-peptide, small-
molecular PSA-specific inhibitors with
a
homo-
phthalimide or a quinazolinedione skeleton derived
from thalidomide (1), including N-(2,6-diethylphe-
nyl)homophthalimide (PIQ-22, 2), 3-(2,6-diethylphe-
nyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 3) and 1-
methyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione (MPAQ-22, 4) (Fig. 1).9À16 They are all potent
PSA-specific non-competitive inhibitors with IC50
values of 3–8 mM (Table 1). The potencies of these
inhibitors are comparable to those of bestatin and acti-
nonin (competitive inhibitors).9À16 By employing these
PSA inhibitors, we identified possible roles of the
enzyme in cell mobility/invasion/apoptosis.11À13,16,17
Figure 1. Structures of thalidomide (1), PSA-specific inhibitors (PIQ-
22: 2, PAQ-22: 3, MPAQ-22: (4) derived from thalidomide, and the
designed fluorescent bioprobes (ANTAQ: 5, DAMPAQ-22: 6).
*Corresponding author. Tel.: +81-3-5841-7847; fax: +81-3-5841-
8495; e-mail: hashimot@iam.u-tokyo.ac.jp
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00845-4

84
H. Kakuta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 83–86
For derivatization of PAQ-22 (3)/MPAQ-22 (4) to
fluorescent analogues, two strategies were applied on
the basis of the results of our previous structure–activity
relationship studies,9À16 that is (i) derivatization of the
2,6-diethylphenyl group, which is critical for potent
PSA-inhibiting activity, to a fluorescent 9-anthracenyl
group, and (ii) introduction of a fluorescent dansyl
group into the 2,6-diethylphenyl group at the 4-posi-
tion, which was not expected to drastically affect the
activity. Thus, we designed ANTAQ (5) and DAM-
PAQ-22 (6) (Fig. 1).
(vide infra), and showed no apparent cytotoxicity within
the concentration range used in the experiments, as far as
could be judged by optical and fluorescence microscopy.
As is generally the case for fluorescent compounds, the
fluorescence intensity of both ANTAQ (5) and DAM-
PAQ-22 (6) was much higher under hydrophobic con-
ditions than under the hydrophilic conditions.20À22 By
making use of this characteristic, ANTAQ (5) and
DAMPAQ-22 (6) could be used as fluorescent probes to
detect PSA without a procedure to separate the free
form of the probe from the bound form (F/B
separation),20À22 that is mixing ANTAQ (5) or DAM-
PAQ-22 (6) with a sample solution containing PSA
resulted in an increase of the fluorescence intensity.
Moreover, the increase of fluorescence intensity was
diminished by addition of non-fluorescent inhibitors,
including PIQ-22 (2), PAQ-22 (3) and MPAQ-22 (4)
(details will be published elsewhere). The results sug-
gested that (i) the increased fluorescence intensity is due
to the specific binding to PSA of ANTAQ (5) and
DAMPAQ-22 (6), (ii) they bind specifically at the same
binding site as PIQ-22 (2)/PAQ-22 (3)/MPAQ-22 (4),
and (iii) ANTAQ (5) and DAMPAQ-22 (6) can be used
as fluorescent probes for visualization of PSA.
ANTAQ (5) was prepared by condensation of 9-amino-
anthracene and methyl anthranilate in the presence of
triphosgene by the method described previously (Fig. 2).15
DAMPAQ-22 (6) was prepared by condensation of
dansyl chloride and 1-methyl-3-(2,6-diethyl-4-amino-
phenyl)-2,4(1H,3H)-quinazolinedione, which was itself
prepared by condensation of 2,6-diethyl-4-nitroaniline
with methyl anthranilate in the presence of triphosgene
followed by reduction of the nitro group to an amino
group (Fig. 2). The structures of the prepared com-
pounds were confirmed by 1H NMR, mass spectro-
scopy, and elemental analysis, which gave appropriate
analytical values.18,19
As expected, ANTAQ (5) and DAMPAQ-22 (6) showed
potent PSA-inhibiting activity and possessed high
fluorescence intensity.18,19 Their PSA-inhibiting activity
was comparable to those of the mother compounds,
PAQ-22 (3) and MPAQ-22 (4), with IC50 values of 2.9
and 4.6 mM, respectively (Table 1). They are inactive
toward APN and dipeptidylpeptidase type IV, as are
PAQ-22 (3), MPAQ-22 (4) and PIQ-22 (2), indicating
that they are specific to PSA. Lineweaver–Burk plot ana-
lysis (Fig. 3) indicated that ANTAQ (5) and DAMPAQ-
22 (6) act as non-competitive inhibitors, as do PAQ-22
(3), MPAQ-22 (4) and PIQ-22 (2).11À16 In addition, they
are easily incorporated into living human monocytic
cells MOLT-4 under the general cell culture conditions
We first checked the applicability of ANTAQ (5) and
DAMPAQ-22 (6) as fluorescent bioprobes to visualize
PSA in living cells by the use of four types of cells, that
Table 1. PSA-inhibitory activity of compounds 2–6
Compd
Inhibition
IC50, mMa
Compd
Inhibition
IC50, mMa
PIQ-22: 2
PAQ-22: 3
MPAQ-22: 4
7.8
3.8
3.4
ANTAQ: 5
DAMPAQ-22: 6
2.9
4.6
aPSA-inhibitory activity was assayed by the use of l-Ala-AMC with
MOLT-4.9À16
Figure 3. Lineweaver–Burk plot analysis of PSA inhibition by
(a) ANTAQ (5) and (b) DAMPAQ-22 (6).
Figure 2. Synthesis of ANTAQ (5) and DAMPAQ-22 (6).

H. Kakuta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 83–86
85
or weakly expressing PSA) were not stained or were
only weakly stained (no localized staining was
observed). The ratio of the fluorescence intensity of
MOLT-4 and HL-60 stained by DMAPAQ-22 (6) was
11:3. The results indicated that both ANTAQ (5) and
DAMPAQ-22 (6) are superior fluorescent bioprobes for
visualization of PSA in living cells. The fluorescent
microscopic observation of MOLT-4 cells treated with
ANTAQ (5) and DAMPAQ-22 (6) suggested a specific
localization pattern of PSA in the cells. Typical images
are shown in Figure 4, though the localization pattern is
not entirely uniform among the cells.
Figure 4. Fluorescent microscopic images of MOLT-4 cells treated
with 10 mM (a) ANTAQ (5) or (b) DAMPAQ-22 (6) for 10 min at
37 ꢀC. U-MNUA2 filter (Ex/Em: 360/405 nm) and U-WU2 filter (Ex/
Em: 360/510 nm) were used for panels (a) and (b), respectively.
Comparison of the photographs of fluorescence images
(Fig. 4) with optical microscopic images (not shown) of
MOLT-4 cells with or without Wright–Giemsa staining
suggested that PSA exists in the cytosolic fraction, not
in the nucleus, which is in accordance with our previous
results.9À16 Moreover, PSA seemed to be localized at the
cleft or groove of karyolobism (lobulated nucleus) in the
cell.
In order to gain further information concerning the
distribution/localization of PSA in living cells, fluor-
escent staining with other probes, including Dil C16(3)
[which has been established to stain endoplasmic retic-
ulum (ER), Molecular Probes Co. Ltd.] and 5-DTAF
(which has been established to stain tubulin, Molecular
Probes Co. Ltd.), was carried out (Fig. 5).
In the overlaid images in Figure 5 (the right images in
panels A–C), areas colored with yellow/orange indicate
co-localization of PSA (colored green in the middle
images in panels A–C) with ER (colored with red in the
left images in panels A and B) or tubulin (colored with
purple in the left image in the panel C). At first glance,
PSA seems to co-localize with both ER and tubulin.
However, the yellow areas in the overlaid images in
panels A and B are much smaller than that observed in
the overlaid image in panel C. The results suggest that
PSA is distributed in a manner which is more similar to
tubulin than to ER. Further investigation of the effects
of PSA inhibitors on cell mobility/invasion and
apoptosis is scheduled.
Figure 5. Fluorescence microscopic images of MOLT-4 cells treated
with ANTAQ (5), DAMPAQ (6), Dil C16(3) and 5-DTAF. (A) Dil
C16(3) (1 mM, ER staining), ANTAQ (5, 1 0mM, PSA staining), for 10
min at 37 ꢀC, and their overlay. (B) Dil C16(3) (1 mM, ER staining),
DAMPAQ-22 (6, 1 0mM, PSA staining), for 10 min at 37 ꢀC, and their
overlay. (C) 5-DTAF (1 mM, tubulin staining), ANTAQ (5, 1 0mM,
PSA staining). White bars in the overlay panels indicate 20 mm. Filters
used for ANTAQ (5) and DAMPAQ-22 (6) were the same as Figure 4.
For Dil C16(3) and 5-DTAF, U-WIG2 filter (Ex/Em: 549/565 nm) was
used.
In conclusion, PSA-specific fluorescent bioprobes,
ANTAQ (5) and DAMPAQ-22 (6), were prepared by
structural modification of our potent PSA-specific inhi-
bitors. The usefulness of these bioprobes for visualiza-
tion of PSA in living cells was demonstrated. Because
these bioprobes also possess potent PSA-inhibiting
activity, they should be useful tools to investigate so far
unknown physiological role(s) of PSA.
is, human monocytic cell line MOLT-4, which is known
to express PSA,9À16 human leukemia cell line HL-60,
which lacks or only weakly expresses PSA,12,16 and
COS-1cells transfected or not transfected with a PSA
expression vector. As expected, MOLT-4 and PSA-
transfected COS-1cells (cells expressing PSA) were effi-
ciently stained by ANTAQ (5) or DAMPAQ-22 (6),
while HL-60 and non-treated COS-1cells (cells lacking
Acknowledgements
The work described in this paper was partially sup-
ported by Grants-in-Aid from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan.
The authors are grateful to Dr. Akimitsu, Mr. Miyagi, and
Mr. Ogawa (Olympus Co. Ltd.) for their technical support
in obtaining fluorescence microscopic photographs.

86
H. Kakuta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 83–86
17. Kagechika, H.; Komoda, M.; Fujimoto, Y.; Koiso, Y.;
References and Notes
Takayama, H.; Kadoya, S.; Miyata, K.; Kato, F.; Kato, M.;
Hashimoto, Y. Biol. Pharm. Bull. 1999, 22, 1010.
1. Hersh, L. B.; McKelvy, J. F. J. Neurochem. 1981, 36, 171.
2. McLellan, S.; Dyer, S. H.; Hersh, L. B. J. Neurochem. 1988,
51, 1552.
3. Johnson, G. D.; Hersh, L. B. Arch. Biochem. Biophys. 1990,
276, 305.
4. Rawlings, N. D.; Barrett, A. J. Biochem. J. 1993, 290, 201.
5. Tobler, A. R.; Constam, D. B.; Schmitt-Graff, A.; Mal-
ipiero, U.; Schlabach, R.; Fontana, A. J. Neurochem. 1997, 68,
889.
6. Costam, D. B.; Tobler, A. R.; Rensing-Ehl, A.; Kelmer, I.;
Hersh, L. B.; Fontana, A. J. Biol. Chem. 1995, 270, 26931.
7. Bauer, M. O.; Nanda, I.; Beck, G.; Schmid, M.; Jacob, F.
Cytogenet. Cell Genet. 2001, 92, 221.
8. Osada, T.; Ikegami, S.; Takiguchi-Hayashi, K.; Yamazaki,
Y.; Katoh-Fukui, Y.; Higashikanagawa, T.; Sasaki, Y.;
Takeuchi, T. J. Neurochem. 1999, 19, 6068.
9. Hashimoto, Y. Bioorg. Med. Chem. 2002, 10, 461.
10. Hashimoto, Y. Curr. Med. Chem. 1998, 5, 163.
11. Shimazawa, R.; Takayama, H.; Fujimoto, Y.; Komoda,
M.; Dodo, K.; Yamasaki, Y.; Shirai, R.; Koiso, Y.; Miyata,
K.; Kato, F.; Kato, M.; Miyachi; Hashimoto, Y. J. Enzyme
Inhibit. 1999, 14, 259.
12. Takahashi, H.; Komoda, M.; Kakuta, H.; Hashimoto, Y.
Yakugaku Zasshi 2000, 120, 909.
13. Kakuta, H.; Takahashi, H.; Sou, S.; Kita, T.; Nagasawa, K.;
Hashimoto, Y. Recent Res. Develop. Med. Chem. 2001, 1, 1 89.
14. Miyachi, H.; Kato, M.; Kato, F.; Hashimoto, Y. J. Med.
Chem. 1998, 41, 263.
18. 3-(9-Anthracenyl)-2,4(1H,3H)-quinazolinedione (ANTAQ:
5) Pale yellow cubes from EtOAc; mp >300 ꢀC; 1H NMR
(500MHz, DMSO-d6) d: 16.60 (s, 1H), 8.78 (s, 1H), 8.20 (d, 2H,
J=7.5 Hz), 7.98 (d, 1H, J=7.5 Hz), 7.82 (d, 2H, J=7.5 Hz),
7.80 (t, 1H, J=7.5 Hz), 7.57 (t, 2H, J=7.5 Hz), 7.51(t, 2H,
J=7.5 Hz), 7.38 (d, 1H, J=7.5 Hz), 7.30 (t, 1H, J=7.5 Hz);
FAB-MS m/z: 339 (M+H)+. Anal. calcd for C22H14N2O2 C,
78.09; H, 4.17; N, 8.28. Found: CHN. Wavelengths of the max-
imum fluorescence intensity (Ex/Em): 360/405 nm (benzene),
370/405 nm (CH2Cl2), 360/405 nm (MeOH).
19. 3-(2,6-Diethyl-4-dansylaminophenyl)-1-methyl-2,4(1H,3H)-
quinazolinedione (DAMPAQ-22: 6). Pale green cubes from
MeOH; mp 244 ꢀC; H NMR (500 MHz, CDCl3) d: 8.50 (d,
1
1H, J=8.0 Hz), 8.39 (d, 1H, J=8.0 Hz), 8.25 (d, 1H, J=8.0
Hz), 8.24 (d, 1H, J=8.0 Hz), 7.74 (td, 1H, J=8.0, 1.5 Hz),
7.55 (t, 1H, J=8.0 Hz), 7.46 (d, 1H, J=8.0 Hz ), 7.44 (s, 1H),
7.29 (t, 1H, J=8.0 Hz), 7.28 (t, 1H, J=8.0 Hz), 7.17 (d, 1H,
J=8.0 Hz), 6.68 (s, 2H), 3.66 (s, 3H), 2.87 (s, 6H), 2.23 (q, 2H,
J=7.5 Hz), 2.24 (q, 2H, J=7.5 Hz), 0.91(t, 6H, J=7.5 Hz);
FAB-MS m/z: 556 (M)+, 557 (M+H)+. Anal. calcd for
C31H32N4O4: C, 66.88; H, 5.79; N, 10.06. Found: C, 66.72; H,
5.85; N, 10.04. Wavelengths of the maximum fluorescence
intensity (Ex/Em): 320/490 nm (benzene), 320/510 nm
(CH2Cl2), 320/510 nm (MeOH).
20. Shimazawa, R.; Hibino, S.; Mizoguchi, H.; Hashimoto,
Y.; Iwasaki, S.; Kagechika, H.; Shudo, K. Biochem. Biophys.
Res. Comm. 1991, 180, 249.
15. Kakuta, H.; Koiso, Y.; Takahashi, H.; Nagasawa, K.;
Hashimoto, Y. Heterocycles 2001, 55, 1433.
21. Hashimoto, Y.; Shimazawa, R. Clin. Dermatol. 1993, 5,
585.
16. Komoda, M.; Kakuta, H.; Takahashi, H.; Fujimoto, Y.;
Kadoya, S.; Kato, F.; Hashimoto, Y. Bioorg. Med. Chem.
2001, 9, 121.
22. Sawada, T.; Kato, Y.; Kobayashi, H.; Hashimoto, Y.;
Watanabe, T.; Sugiyama, Y.; Iwasaki, S. Bioconjugate Chem.
1993, 4, 284.