Design of fluorogenic probes and fluorescent-tagged inhibitors for γ-glutamyl cyclotransferase

Article abstract of DOI:10.1002/psc.2984

A tumor-related protein, human chromosome 7 ORF 24 (C7orf24), is involved in regulation of the glutathione homeostasis cycle as a γ-glutamyl cyclotransferase (GGCT). The singular substrate preference of this enzyme had long hampered its chemical probe development. That is, substrate of GGCT is definitely ‘γ-Glu-Xaa’, where Xaa is an L-α-amino acid. Based on the structure of substrates and GGCT fluorogenic probes, LISA-4/101, we succesfully developed a fluorescent-tagged inhibitor, gKFA. These chemical tools will assist cancer-related researches in the future.

Full text of DOI:10.1002/psc.2984

Special Issue Article
Received: 16 November 2016
Revised: 25 January 2017
Accepted: 28 January 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2984
Design of fluorogenic probes and
fluorescent-tagged inhibitors for γ-glutamyl
cyclotransferase
Taku Yoshiya,a* Hiromi Ii,b Shugo Tsuda,a Masayoshi Mochizuki,a
Susumu Kageyamac and Tatsuhiro Yoshikib
A tumor-related protein, human chromosome 7 ORF 24 (C7orf24), is involved in regulation of the glutathione homeostasis cycle as
a γ-glutamyl cyclotransferase (GGCT). The singular substrate preference of this enzyme had long hampered its chemical probe
development. That is, substrate of GGCT is definitely ‘γ-Glu-Xaa’, where Xaa is an L-α-amino acid. Based on the structure of sub-
strates and GGCT fluorogenic probes, LISA-4/101, we succesfully developed a fluorescent-tagged inhibitor, gKFA. These chemical
tools will assist cancer-related researches in the future. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: activity-based probe; cancer; fluorescence; GGCT; inhibitor
Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Nacalai
Tesque, Inc. (Kyoto, Japan), Watanabe Chemical Industries, Ltd.
Introduction
A tumor-related protein, human chromosome 7 ORF 24 (C7orf24), is
involved in regulation of the glutathione homeostasis cycle as a
γ-glutamyl cyclotransferase (GGCT) [1–3]. GGCT specifically converts
γ-glutamyl amino acids (γ-Glu-Xaa) into pyroglutamic and
corresponding amino acids. In contrast, GGCT does not recognize
γ-glutamyl peptides (Figure 1). Although its definite role remains
unclear, GGCT is thought to accelerate cancer progression, and its
inhibitor should function as an anti-cancer drug [4–6]. Specifically,
systemic administration of small interfering RNA (siRNA) of GGCT
could retard tumor growth without toxicity to normal tissues [7].
Thus, GGCT is a promising target for cancer treatment. Since the
discovery of C7orf24 in 2007 [8], we studied GGCT using its
antibody and siRNA. Although the antibody against GGCT can
detect C7orf24, it does not reflect GGCT activity. Additionally, the
siRNA against C7orf24 downregulates the expression of C7orf24;
however, the effect of siRNA may not be attributed to downregula-
tion of GGCT activity. Thus, to gain further understanding of GGCT,
activity-based probe and GGCT inhibitor are indispensable [9].
Specifically, a GGCT inhibitor that possesses fluorescence should
be beneficial for chemical biology-oriented research regarding
drug delivery.
(Hiroshima, Japan), and Merck KGaA (Darmstadt, Germany).
Preparation of LISA-4/101 is described in References [10,11],
respectively. Analytical HPLC was performed on a Shimadzu liquid
chromatograph Model LC-2010C (Kyoto, Japan) with a YMC-ODS
AA12S05-1546WT (4.6 × 150 mm) and the following solvent
systems: (i) 0.1% TFA in H2O and (ii) 0.1% TFA in CH3CN at a flow rate
of 1 ml minÀ1 (40 °C) with detection at 220 nm. Preparative HPLC
was carried out on a Shimadzu liquid chromatograph Model
LC-8A or LC-20AP (Kyoto, Japan) with a YMC-ODS AA12S05-
2530WT (30 × 250 mm) and the following solvent systems:
(i) 0.1% TFA in H2O and (ii) 0.1% TFA in CH3CN at a flow rate of
20 ml minÀ1 (room temperature) with detection at 220 nm. Mass
spectra were observed with an Agilent G1956B LC/MSD detector
1
using an Agilent 1100 series HPLC system. H NMR spectra were
recorded on a JEOL-ECX400 spectrometer (Tokyo, Japan) with the
residual solvent peak as an internal reference.
In this context, we have designed the fluorogenic probe LISA-4
[10] and LISA-101 [11] with the chemical and enzymatic ligand acti-
vation system based on the structure of native substrates. Prior to
this, the singular substrate preference of GGCT had long hampered
its chemical probe development. Additionally, we also designed a
novel fluorescent-tagged inhibitor, gKFA. Herein, we report how
these chemical biology-oriented compounds were designed.
*
Correspondence to: Taku Yoshiya, Peptide Institute, Inc., Ibaraki, Osaka 567-0085,
Japan. E-mail: t.yoshiya@peptide.co.jp
a Peptide Institute, Inc., Ibaraki, Osaka 567-0085, Japan
b Department of Clinical Oncology, Kyoto Pharmaceutical University, Kyoto, Kyoto
607-8412, Japan
c
Department of Urology, Shiga University of Medical Science, Otsu, Shiga
520-2192, Japan
Abbreviations: A2bu, (S)-2,4-diaminobutanoic acid; A2pr, (S)-2,3-
Materials and Methods
diaminopropanoic acid; C7orf24, human chromosome
7 ORF 24; GGCT,
γ-Glutamyl cyclotransferase; gKFA, glutaryl-Lys(NBD); LISA-101, γ-Glu-A2bu(N-Et,
CO-resorufin); LISA-4, γ-Glu-Ser(CO-4-methylumbelliferone); NBD-Cl, 4-chloro-7-
nitro-2,1,3-benzoxadiazole
All reagents and solvents were obtained from the Peptide Institute,
Inc. (Osaka, Japan), Wako Pure Chemical Industries, Ltd. (Osaka,
J. Pept. Sci. 2017
Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

YOSHIYA ET AL.
Figure 1. GGCT reaction: γ-Glu-Xaa is a substrate, but γ-Glu-peptide is not a substrate. Important carboxylic acid moiety is marked by the red color.
γ-Glu-D-Lys(Z)
J = 6.6 Hz, 2H), 2.42–2.22 (m, 2H), 2.05–1.87 (m, 2H), 1.73–1.48 (m,
2H), 1.45–1.19 (m, 4H); ESI-MS Calcd. (M+H)+: 410.2, Found: 410.1.
H-D-Lys(Z)-OH (0.20 g, 0.71 mmol), Boc-Glu(ONSu)-OtBu (0.27 g,
0.68 mmol), and triethylamine (0.11 ml, 0.78 mmol) were dissolved
in DMF (2 ml) and stirred overnight at room temperature. Then,
ethyl acetate (EtOAc) was added to the reaction mixture, and the
organic layer was washed successively with 1 N HCl and brine.
Boc-Glu[D-Lys(Z)-OH]-OtBu was condensed in vacuo and used
without further purification.
Glutaryl-Lys(NBD) (gKFA)
H-Lys(Z)-OBzl•HCl (1.50 g, 3.69 mmol), glutaric anhydride (926 mg,
8.12 mmol), and diisopropylethylamine (DIEA, 2.41 ml, 13.9 mmol)
were dissolved in DMF (8 ml) and stirred for 4 h at room tempera-
ture. The remaining glutaric anhydride was then quenched by
addition of N,N-dimethyl-1,3-propanediamine (690 μl, 5.54 mmol).
EtOAc was added to the reaction mixture, and the organic layer
was washed successively with 1 N HCl and brine. Glutaryl-Lys(Z)-
OBzl was condensed in vacuo and used without further purification.
To the AcOH (10 ml) solution of glutaryl-Lys(Z)-OBzl, 5% Pd/C
(300 mg) was added, and the mixture was stirred vigorously under
an H2 atmosphere overnight. After filtration, glutaryl-Lys was
condensed in vacuo and used without further purification.
To the MeOH (13 ml)-sat. NaHCO3 aq. (15 ml) solution of
glutaryl-Lys, 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl, 1.84 g,
9.22 mmol) was added. The solution was stirred overnight at
65 °C. After acidification by addition of AcOH, the mixture was
purified by RP-HPLC (H2O-CH3CN system containing 0.1% TFA).
The desired fractions were collected and lyophilized to afford the
title compound as an orange amorphous powder (230 mg,
Boc-Glu[D-Lys(Z)-OH]-OtBu was dissolved in CF3COOH (10 ml)
containing H2O (0.3 ml), and the mixture was stirred for 1 h at room
temperature. After condensation in vacuo, the residue was purified
by RP-HPLC (H2O-CH3CN system containing 0.1% TFA). The desired
fractions were collected and lyophilized to afford the title com-
pound as an amorphous powder (0.067 g, 0.13 mmol). Purity:
1
98.4% (220 nm); H NMR (DMSO, 400 MHz) δ 8.21 (d, J = 7.8 Hz,
1H), 7.39–7.26 (m, 5H), 7.23 (t, J = 5.7 Hz, 1H), 4.99 (s, 2H), 4.17–
4.05 (m, 1H), 3.74 (t, J = 6.4 Hz, 1H), 2.96 (q, J = 6.6 Hz, 2H), 2.41–
2.21 (m, 2H), 2.05–1.84 (m, 2H), 1.76–1.47 (m, 2H), 1.47–1.18 (m,
4H); ESI-MS Calcd. (M+H)+: 410.2, Found: 410.1.
γ-Glu-N-Me-Lys(Z)
The title compound was synthesized in a similar manner to γ-Glu-D-
Lys(Z). Yield: 0.064 g, 0.12 mmol (starting from 0.65 mmol of Glu
1
1
543 μmol). Purity: 99.0% (220 nm); H NMR (DMSO-d6 containing
derivative); Purity: 95.8% (220 nm); H NMR (DMSO, 400 MHz) δ
5% CF3COOD, 400 MHz) δ 8.48 (d, J = 8.7 Hz, 1H), 6.38 (d, J = 8.7
Hz, 1H), 4.15 (dd, J = 9.1, 4.6 Hz, 1H), 3.53–3.35 (m, 2H), 2.22–2.08
(m, 4H), 1.79–1.52 (m, 4H), 1.48–1.33 (m, 2H); ESI-MS Calcd.
(M+H)+: 424.1, Found: 424.1.
7.38–7.19 (m, 6H), 4.98 (s, 2H), 4.87 (dd, J = 10.8, 4.8 Hz, 0.8H*),
4.41–4.32 (m, 0.2H*), 3.94–3.83 (m, 1H), 3.05–2.91 (m, 2H), 2.82 (s,
2.3H*), 2.65 (s, 0.7H*), 2.61–2.36 (m, 2H), 2.12–1.56 (m, 4H),
1.53–1.29 (m, 2H), 1.28–1.04 (m, 2H) [Rotamers* (77:23) at N-Me
amide were observed.]; ESI-MS Calcd. (M+H)+: 424.2, Found: 424.2.
Glutaryl-Lys(NBD) dimethyl ester (Me-gKFA-Me)
β-Asp-Lys(Z)
To the solution of glutaryl-Lys(NBD) (70.0 mg, 165 μmol) in
CH2Cl2-MeOH (1:1, 7 ml), was added the hexane solution of
trimethylsilyldiazomethane (0.6 M, 1.5 ml, 0.9 mmol) at room
temperature for 1 h to afford the dimethyl ester and monomethyl
ester mixture of glutaryl-Lys(NBD) (ca 6 : 4). The remaining
trimethylsilyldiazomethane was then quenched by addition of
AcOH (60 μl). The mixture was condensed in vacuo, and the residue
was dissolved in DMF and purified by RP-HPLC (H2O-CH3CN system
containing 0.1% TFA). The desired fractions were collected
and lyophilized to afford the title compound as an orange
amorphous powder. Yield: 36.8 mg, 81.5 μmol. Purity 98.7%
(220 nm); 1H NMR (DMSO-d6 containing 5% CF3COOD, 400 MHz) δ
8.49 (d, J = 8.7 Hz, 1H), 6.38 (d, J = 8.7 Hz, 1H), 4.20 (dd, J = 9.1,
5.0 Hz, 1H), 3.58 (s, 3H), 3.55 (s, 3H), 3.50–3.38 (m, 2H), 2.26
(t, J = 7.3 Hz, 2H), 2.12 (t, J = 7.1 Hz, 2H), 1.78–1.55 (m, 6H),
1.47–1.32 (m, 2H); ESI-MS Calcd. (M+H)+: 452.2, Found: 452.2.
The title compound was synthesized in a similar manner to γ-Glu-D-
Lys(Z). Yield: 0.070 g, 0.13 mmol (starting from 0.67 mmol of Asp
1
derivative); Purity: >99.5% (220 nm); H NMR (DMSO, 400 MHz) δ
8.50 (d, J = 7.3 Hz, 1H), 7.38–7.20 (m, 6H), 4.99 (s, 2H), 4.21–4.11
(m, 1H), 4.00–3.90 (m, 1H), 2.96 (q, J = 6.4 Hz, 2H), 2.79 (dd,
J = 16.9, 4.6 Hz, 1H), 2.69 (dd, J = 16.5, 6.9 Hz, 1H), 1.76–1.48 (m,
2H), 1.45–1.18 (m, 4H); ESI-MS Calcd. (M+H)+: 396.2, Found: 396.1.
γ-Glu-Lys(Z)
The title compound was synthesized in a similar manner to γ-Glu-D-
Lys(Z). Yield: 0.12 g, 0.23 mmol (starting from 0.36 mmol of Glu
1
derivative); Purity: >99.5% (220 nm); H NMR (DMSO, 400 MHz) δ
8.24 (d, J = 7.8 Hz, 1H), 7.39–7.26 (m, 5H), 7.22 (t, J = 5.7 Hz, 1H),
4.99 (s, 2H), 4.16–4.06 (m, 1H), 3.84 (t, J = 6.4 Hz, 1H), 2.96 (q,
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FLUOROGENIC PROBES AND FLUORESCENT-TAGGED INHIBITORS FOR GGCT
GGCT reaction for substrate investigation
Phosphate buffered solution (pH 8, 100 mM) containing a putative
substrate (0.1 mM) and recombinant GGCT (6 μg/ml) were left at
room temperature. After 18 min, mixture was directly analyzed by
RP-HPLC (UV). Compound decomposed by GGCT less than 1%
was considered as ‘non-substrate’. In the same condition, γ-Glu-
Lys(Z) is decomposed ca. 20%.
GGCT reaction for inhibitor investigation
HEPES buffered solution (pH 8, 100 mM) containing a putative
inhibitor (83 μM), LISA-101 (25 μM), and recombinant GGCT
(48 μg/ml) were left at room temperature. After 50 min, mixture
was directly analyzed by RP-HPLC (UV). The inhibitory activity (%)
was calculated based on the decomposition of LISA-101 in the
absence of the putative inhibitor.
Uptake in cells of the fluorescence-labeled GGCT inhibitors
NIH3T3 cells were seeded at 3 × 104 cells/well in collagen coated
eight-well glass chamber slide. The next day, cells were treated with
100 μM of gKFA or Me-gKFA-Me. After 15 min incubation, cells were
washed with PBS and observed with a fluorescence microscope.
Figure 2. In order to evaluate the scope of the substrate reactivity of GGCT,
these compounds were synthesized and applied to the GGCT assay. Among
them, only γ-Glu-Lys(Z) was a substrate for GGCT.
Figure 3. (A) Structures of LISA-4/101. (B and C) Enzymatic reactions and subsequent chemical reactions of LISA-4/101.
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YOSHIYA ET AL.
Table 1. Km and kcat values of LISA-4/101 and γ-Glu-Lys(Z)
contains a latent fluorophore and releases an intact fluorophore
upon enzymatic cleavage, γ-Glu-Xaa should become a GGCT
fluorogenic probe. Based on this assumption, as a proof-of-concept
probe, we designed a dipeptide-type compound γ-Glu-Ser(CO-4-
methylumbelliferone) (LISA-4). LISA-4 consists of γ-glutamic acid
and the Ser derivative that possesses the carbonate bond-linked
fluorophore 4-methylumbelliferone (MU) on its side chain
(Figure 3). Fluorescence of MU is suppressed by O-acylation. Addi-
tionally, when an α-amino group of Ser is liberated by GGCT, the
Ser derivative releases the intact fluorophore ‘MU’ via an intramolec-
ular cleavage reaction at the carbonate bond with cyclic lactam for-
mation. As a result, fluorescence was regained from LISA-4 when the
probe is treated with GGCT. Then, we designed γ-Glu-A2bu(N-Et, CO-
resorufin) (LISA-101) as a stable fluorogenic probe (Figure 3). In LISA-
101, the connection of the fluorophore was replaced by an N-alkyl
urethane structure, which is known to be more stable than the car-
bonate bond used in LISA-4. For this purpose, Ser in the structure of
LISA-4 was replaced with A2bu [12]. Eventually, LISA-101 was stable
under physiological conditions and released an intact fluorophore
resorufin in the presence of GGCT, while LISA-4 is stable under pH
6.5 but not at pH 7.5 or higher (e.g. half-life = ca. 4.4 h at pH 8). More-
over, fluorophore was changed to resorufin, which possesses a rela-
tively long wavelength fluorescence emission. As a result, LISA-101
could be applied to a cell-based assay using a plate reader. Km and
pH
Km (mM)
kcat (mM/min/μg)
kcat/Km
LISA-4
6.5
8
0.38
0.56
0.28
0.32
0.0067
0.013
0.14
0.018
0.023
0.50
LISA-101
γ-Glu-Lys(Z)
γ-Glu-Lys(Z)
6.5
8
0.21
0.66
Km and kcat values were evaluated by the method similar to that
reported in Reference [10].
Results and Discussion
In order to design a GGCT fluorogenic probe and fluorescent-
tagged inhibitor, we focused on the structures of the native sub-
strates. So far, γ-glutamyl amino acids are known as substrates for
GGCT. In contrast, γ-glutamyl peptide is not a substrate. These
results clearly indicate the importance of an α-carboxyl group at
the P1’ site for enzyme substrate interaction. Thus, we screened
several compounds possessing a carboxyl group at the P1’ site in
the enzyme assay.
To evaluate scope of the substrate of GGCT, we designed many
compounds and applied to GGCT. As shown in Figure 2, γ-Glu-pNA
derivatives [10] containing carboxyl groups that mimic an α-
carboxyl group and γ-Glu-Lys(Z) derivatives (N-methylated, D-Lys
and β-Asp) were evaluated. Unfortunately, none of them was sub-
strate for GGCT. Meanwhile, γ-Glu-Lys(Z) was a GGCT substrate, sug-
gesting that a relatively large side chain is acceptable at the P1’ site.
Judging from these results, we concluded that the probe should
be γ-Glu-Xaa, where Xaa is restricted to an L-α-amino acid. When Xaa
kcat values were evaluated for LISA-4/101, and we compared these
values to the native-type substrate γ-Glu-Lys(Z). The results are sum-
marized in Table 1. The Km values of LISA-4/101 were not very differ-
ent from the native-type substrate; kcat values of LISA-4/101 were,
however, ca. 20 times smaller than that of the native-type substrate.
These results may be because the bulky side chain on the P1’ site
prevented formation of the transition states in the enzyme-
substrate complex.
Figure 4. Docking images and speculative interactions with GGCT; (A) The native-type ligand, γ-Glu-Cys, and (B) the GGCT inhibitor, glutaryl-Ala.
Crystallographic data were retrieved from PDB (2PN7).
Table 2. Structures and inhibitory activities of glutaryl-Ala, gKFA, and Me-gKFA-Me
Glutaryl-Ala
54
gKFA
53
Me-gKFA-Me
Inhibitory activity (%)
<5
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FLUOROGENIC PROBES AND FLUORESCENT-TAGGED INHIBITORS FOR GGCT
Figure 5. Cell permeability of fluorescent-tagged inhibitors was evaluated using a fluorescence microscope.
Based on the successful development of a GGCT probe, we
designed a fluorescent-tagged inhibitor based on the structure
of a known inhibitor, glutaryl-Ala [13,14]. As described earlier,
GGCT strongly interacts with substrates at the α-carboxyl group
of the P1’ site. We assumed that the interaction with Arg30 of
GGCT is important for the inhibitor too (Figure 4). In contrast, a
side chain of an amino acid at the P1’ site of substrates is not re-
stricted to a native structure. Thus, we simply introduced a
fluorophore (NBD) at the side chain of the P1’ site to design
the fluorescence-tagged inhibitor gKFA [glutaryl-Lys(NBD)]. Inhib-
itory activities were evaluated using LISA-101 and summarized in
Table 2. As expected, inhibitory activity of gKFA was similar to
that of glutaryl-Ala. Simultaneously, IC50 value of glutaryl-Ala
was measured as 0.1 mM [15]. In contrast, its dimethyl ester de-
rivative (Me-gKFA-Me), as a putative prodrug, did not show in-
hibitory activity, indicating the importance of the carboxylic
acid. Additionally, we evaluated cell permeability of these com-
pounds using NIH-3T3 cells. As shown in Figure 5, the parent
compound gKFA did not enter the cells. In contrast, as had been
expected, Me-gKFA-Me was efficiently internalized to the cells.
Application of this fluorescence-tagged inhibitor will be pub-
lished elsewhere.
HI and a JSPS KAKENHI Grant numbers 15K10585 and 15K10584
from Japan Society for the Promotion of Science to TatY and SK.
References
1 Orlowsli M, Richman PG, Meister A. Isolation and properties of γ-L-
glutamylcyclotransferase from human brain. Biochemistry 1969; 8:
1048–1055. DOI: 10.1021/bi00831a036.
2 Board PG, Smith JE, Moore K. A simple method to determine γ glutamyl-
cyclotransferase activity in erythrocytes. J. Lab. Clin. Med. 1978; 91:
127–131.
3 Oakley AJ, Yamada T, Liu D, Coggan M, Clark AG, Board PG. The
identification and structural characterization of C7orf24 as γ-Glutamyl
cyclotransferase: an essential enzyme in the gamma-glutamyl cycle.
J. Biol. Chem. 2008; 283: 22031–22042. DOI: 10.1074/jbc.M803623200.
4 Gromov P, Gromova I, Friis E, Timmermans-Wielenga V, Rank F, Simon R,
Sauter G, Moreira JMA. Proteomic profiling of mammary carcinomas
identifies C7orf24, a γ-glutamyl cyclotransferase, as a potential cancer
biomarker. J. Proteome Res. 2010; 9: 3941–3953. DOI: 10.1021/
pr100160u.
5 Hama S, Arata M, Nakamura I, Kasetani T, Itakura S, Tsuchiya H, Yoshiki T,
Kogure K. Prevention of tumor growth by needle-free jet injection of
anti-C7orf24 siRNA. Cancer Gene Ther. 2012; 19: 553–557. DOI: 10.1038/
cgt.2012.31.
6 Takemura K, Kawachi H, Eishi Y, Kitagaki K, Negi M, Kobayashi M,
Uchida K, Inoue J, Inazawa J, Kawano T, Board PG. γ-
Glutamylcyclotransferase as a novel immunohistochemical biomarker
for the malignancy of esophageal squamous tumors. Hum. Pathol.
2014; 45: 331–341. DOI: 10.1016/j.humpath.2013.09.009.
Summary
7 Ran R, Liu Y, Gao H, Kuang Q, Zhang Q, Tang J, Fu H, Zhang Z, He Q.
PEGylated hyaluronic acid-modified liposomal delivery system with
anti-γ-glutamylcyclotransferase siRNA for drug-resistant MCF-7 breast
cancer therapy. J. Pharm. Sci. 2015; 104: 476–484. DOI: 10.1002/
jps.24163.
8 Kageyama S, Iwaki H, Inoue H, Isono T, Yuasa T, Nogawa M, Maekawa T,
Ueda M, Kajita Y, Ogawa O, Toguchida J, Yoshiki T. A novel tumor-related
protein, C7orf24, identified by proteome differential display of bladder
urothelial carcinoma. Proteomics Clin. Appl. 2007; 1: 192–199. DOI:
10.1002/prca.200600468.
We successfully developed a fluorogenic GGCT probe, LISA-101,
while evaluating the scopes and limitations of the substrate reactiv-
ity of the enzyme. Based on these studies, a dipeptide-type inhibi-
tor gKFA was also designed. The GGCT probe and inhibitor shared
the same features: (i) an intact carboxyl group at the P1’ site is im-
portant and (ii) a relatively large side chain structure is acceptable
at the P1’ site. We are now investigating the physiological roles of
GGCT using combinations of these compounds and
antibody/siRNA. Additionally, we are screening small molecule
GGCT inhibitors as anti-cancer drug candidates using LISA-101.
9 Weiss WA, Taylor SS, Shokat KM. Recognizing and exploiting differences
between RNAi and small-molecule inhibitors. Nat. Chem. Biol. 2007; 3:
739–744. DOI: 10.1038/nchembio1207-739.
10 Yoshiya T, Tsuda S, Mochizuki M, Hidaka K, Tsuda Y, Kiso Y, Kageyama S,
Ii H, Yoshiki T, Nishiuchi Y. A fluorogenic probe for γ-glutamyl
cyclotransferase: application of an enzyme-triggered O–to–N Acyl
migration-type reaction. Chembiochem 2013; 14: 2110–2113. DOI:
10.1002/cbic.201300481.
Acknowledgments
We thank Dr Koushi Hidaka (Kobe Gakuin University) for preparing
Figure 4. This work was supported by Takeda Science Foundation to
J. Pept. Sci. 2017
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YOSHIYA ET AL.
11 Yoshiya T, Ii H, Tsuda S, Kageyama S, Yoshiki T, Nishiuchi Y. A GGCT
fluorogenic probe: design, synthesis and application to cancer-related
cells. Org. Biomol. Chem. 2015; 13: 3182–3185. DOI: 10.1039/c5ob00086f.
12 In case of an N-alkyl urethane using A2pr, the probe is not completely
stable under neutral conditions, because nitrogen atom of intramolecu-
lar amide gradually cleaves the N-alkyl urethane (data not shown). In
case of the N-alkyl urethane using A2bu, a similar intramolecular reaction
did not take place.
14 In addition to glutaryl-Ala, β-aminoglutaryl-L-α-aminobutyrate is known
as a competitive inhibitor of GGCT. For details, please refer to the follow-
ing paper: Bridges RJ, Griffith OW, Meister A. L-7-(threo-β-methyl)
glutamyl-L-α-aminobutyrate,
A selective substrate of γ-glutamyl
cyclotransferase. J. Biol. Chem. 1980; 255: 10787–10792.
15 The IC50 value was evaluated using the conditions reported in Ref. 13.
13 Ii H, Yoshiki T, Hoshiya N, Uenishi J. Synthesis and GGCT inhibitory
activity of N-Glutaryl-L-alanine analogues. Chem. Pharm. Bull. 2016; 64:
785–792. DOI: 10.1248/cpb.c16-00167.
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