Design and synthesis of phenolic hydrazide hydrazones as potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.06.065

Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes responsible for catalyzing the formation and degradation of poly(ADP-ribose) (PAR) polymers, respectively. Activation of PARP has been shown to be involved in cell death induced by genotoxic stimuli. On the other hand, genetic disruption of PARG also leads to increased level of cell death by accumulation of PAR. Unlike PARP, where significant medicinal effort has been expended to identify potent inhibitors, PARG has been insufficiently investigated as a molecular therapeutic target. In this study, we report the design, synthesis, and biological evaluation of phenolic hydrazide hydrazones as potent PARG inhibitors. Compounds 3d, 3e, 5d, 5e, 8a, 8b and 8c showed their ability to inhibit the catalytic activity of PARG in vitro with IC₅₀ values of 1.0, 2.1, 3.1, 3.2, 3.1, 2.8 and 1.6 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2014.06.065

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3802–3806
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of phenolic hydrazide hydrazones as potent
poly(ADP-ribose) glycohydrolase (PARG) inhibitors
Rafiqul Islam a, Fumiaki Koizumi b, Yasuo Kodera b, Kengo Inoue c, Tadashi Okawara a,
,
⇑
Mitsuko Masutani d
a Department of Medical Technology, Kumamoto Health Science University, 325 Izumi-machi, Kumamoto 861-5598, Japan
b Sien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
c Pharma Valley Center, 1007 Shimonagakubo, Nagaizumi-machi, Shuntougn, Shizuoka Prefecture 411-4777, Japan
d Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes respon-
sible for catalyzing the formation and degradation of poly(ADP-ribose) (PAR) polymers, respectively.
Activation of PARP has been shown to be involved in cell death induced by genotoxic stimuli. On the other
hand, genetic disruption of PARG also leads to increased level of cell death by accumulation of PAR. Unlike
PARP, where significant medicinal effort has been expended to identify potent inhibitors, PARG has been
insufficiently investigated as a molecular therapeutic target. In this study, we report the design, synthesis,
and biological evaluation of phenolic hydrazide hydrazones as potent PARG inhibitors. Compounds 3d,
3e, 5d, 5e, 8a, 8b and 8c showed their ability to inhibit the catalytic activity of PARG in vitro with IC50
Received 24 January 2014
Revised 18 June 2014
Accepted 21 June 2014
Available online 27 June 2014
Keywords:
Synthesis
Hydrazide-hydrazone
PARG
PARP
values of 1.0, 2.1, 3.1, 3.2, 3.1, 2.8 and 1.6 lM, respectively.
Ó 2014 Elsevier Ltd. All rights reserved.
Inhibitor
Molecular docking
Poly(ADP-ribosyl)ation is a post-translational modification in
which an ADP-ribose unit from nicotinamide adenine dinucleotide
(NAD+) is transferred to specific amino acids of its target proteins.
This modification influences protein–protein interactions and
regulates various cellular processes,1–4 such as cell division, tran-
scription, DNA repair, and different forms of cell death, ranging
from necrosis to apoptosis and autophagy. The synthesis and deg-
radation of poly(ADP-ribose) (PAR) are catalyzed by two types of
enzymes: PAR polymerase (PARP) family of proteins and PAR
glycohydrolase (PARG), respectively.5 PARP1, PARP2 and PARP3
are involved in DNA repair3,4 and PARP inhibitors strongly block
DNA repair.6 Extensive activation of PARP1 by DNA damage has
been specifically linked to the release of apoptosis inducing factor
(AIF) from mitochondria, resulting in NAD+ depletion and cell
death.7 On the other hand, genetic disruption of PARG can rapidly
increase the levels of PAR and cell death, facilitating the release of
mitochondrial proteins through signaling pathways.8–10 PARG
inhibitors do not inhibit PARP1 directly, but instead prevent
PARP1-mediated cell death by slowing the turnover of PAR and
thus slowing NAD+ consumption.11,12
Considering cellular significance, targeting poly(ADP-
ribosyl)ation in the treatment of human diseases, especially cancer,
has attracted considerable attention over the past several years.
For this purpose, PARP inhibitors have been investigated broadly,
since a role for PARP-1 in DNA repair response has been well estab-
lished. To date, chemical inhibitors of PARP enzymes have reached
the first level of clinical application in the therapy of cancer.13–15 In
contrast, due to the lack of significant evidence implicating PARG’s
role in the regulation of cell death and bioavailable inhibitors,
there has been very little progress in identifying small molecule
inhibitors of PARGs. Recently the crystal structures of PARG from
bacterial, protozoan and mammalian sources have been
determined, and the catalytic mechanism of PARG has been
proposed.16–19 PARG comprises an N-terminal regulatory and
targeting domain (A-domain; residues 1–456 in the rat protein),
a central mitochondrial targeting sequence (MTS; residues 457–
472), and a C-terminal catalytic domain (residues 473–972).18
The catalytic domain of rat PARG adopts a bean shaped structure
with a deep central cleft containing the conserved PARG-signature
motif (GGG-X6-8-QEE)18,20 and Tyr791 that contributes strongly to
PARG catalytic efficiency and inhibitor binding.16,21 The glutamate
residues in the signature motif are critical for catalysis, and
mutation of these residues leads to a complete loss of PARG
⇑
Corresponding author. Tel./fax: +81 96 275 2160.
E-mail address: okawara@kumamoto-hsu.ac.jp (T. Okawara).
http://dx.doi.org/10.1016/j.bmcl.2014.06.065
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

R. Islam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3802–3806
3803
Figure 1. Reported PARG inhibitors.
Scheme 2. Synthesis of compounds 8a–h. Reagents and conditions: (a) hydrazine
hydrate, MeOH, reflux, 5–6 h; (b) AcOH, MeOH, reflux, 6–8 h.
activity.16–18,20 In particular, the Glu752 residue could play an
important role in PARG catalytic activity by functioning as a
general acid or base to protonate the 20-OH of the ribose of the
leaving group, and subsequently activating a water molecule for
nucleophilic attack.18 It is noteworthy that PARG is encoded by a
single gene, and it has low cellular abundance (approximately
2000 molecules per cell).22 Progress in understanding the struc-
ture, properties and catalytic mechanism of PARG has created an
opportunity for bioorganic medicinal chemists to design new
anti-cancer drugs that target PARGs.
There are several reports of natural and syntheticPARG inhibitors
that exhibit PARG suppressing activities.11,23–30 However, the use-
fulness of these agents has been limited due to toxicity, lack of spec-
ificity, poor cell permeability and high molecular weights.30–32
Nevertheless, adenosine diphosphate (hydroxymethyl)pyrrolidine-
diol (ADP-HPD),25,26 mono-galloyl glucose,27 RBPI-228 and salicylan-
ilide29 inhibit PARG specifically and help to understand their role
within a wider biological context (Fig. 1). ADP-HPD has been widely
used in the study of PAR in spite of its high cost and lengthy synthetic
Scheme 1. Synthesis of compounds 3a–f and 5a–f. Reagents and conditions: (a) AcOH, MeOH, reflux, 6–8 h; (b) AcOH, MeOH, reflux, 6–8 h.

3804
R. Islam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3802–3806
Table 1
route. Despite these efforts, the design and preparation of potent
PARG inhibitors remain very challenging tasks.
In vitro PARG inhibitory activity of compounds 3a–f, 5a–f and 8a–h
Within this context, we have designed and synthesized small
molecules, phenolic hydrazide hydrazones, and performed
in vitro evaluation of their PARG inhibitory activities. To facilitate
the understanding of PARG binding affinity of the synthesized
compounds, potential binding modes of selected compounds in
the active site of PARG protein were modeled using the X-ray crys-
tal structure of rat PARG (rPARG385, pdb code 3UEK).18
Synthesis of the title compounds 3a–f, 5a–f and 8a–h was
accomplished33 as outlined in Schemes 1 and 2. Condensation of
the appropriate commercially available aryl hydrazides 1 and 4
with the appropriate hydroxybenzaldehydes 2a–f in the presence
of a catalytic amount of acetic acid in methanol under reflux, affor-
ded the corresponding hydrazones 3a–f and 5a–f in quantitative
yields (Scheme 1). The requisite aliphatic dihydrazides 7a–d were
prepared in moderate yields by the reaction of the appropriate
diesters 6a–d with hydrazine hydrate in methanol at elevated
temperatures (Scheme 2). Subsequent reaction of 7a–d with the
appropriate aldehydes 2d,e, under similar conditions used for the
preparation of 3a–f and 5a–f, gave the corresponding second series
Compound
PARG inhibitory activity, IC50 (lM)
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
8a
8b
8c
8d
8e
8f
8g
8h
ADP-HPD
>75
>75
>75
1.0
2.1
9.9
14.8
40.1
19.2
3.1
3.2
18.8
3.1
2.8
1.6
15.4
9.8
8.2
13.9
27.4
0.1
Figure 2. Potential binding modes of compounds 3d, 5d and ADP-HPD within the catalytic domain of rPARG. (A) Alignment of 3d (yellow) and 5d (deep green) within the
active site (electrostatic surface shown). (B) Active site residues showing H-bond interactions (dotted lines) of 3d. (C) Active site residues showing H-bond interactions
(dotted lines) of 5d. (D) Ribbon structure of the rPARG catalytic macrodomain fold (residues 712–918) showing the docking mode of 3d (yellow) and ADP-HPD (magenta) in
the active pocket (green). The tyrosine clasp and Tyr791 acid residue are shown in maroon and cyan, respectively.

R. Islam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3802–3806
3805
of target hydrazones 8a–h in excellent yields. The structures of all
title compounds were confirmed by their physical and spectral
data, with overall yields in the range of 81–95%.33
pound 3d identified its hypothetical binding mode within the
rPARG catalytic domain. As per our preliminary finding, the pheno-
lic hydrazide hydrazone functionality remarkably interacts with
active site of PARG to inhibit its catalytic activity. This finding dem-
onstrates that chemical modification and incorporation of this
functionality in different biologically active scaffolds may lead to
the development of more potent inhibitors of PARG enzyme. Cur-
rently, modification, synthesis and evaluation of PARG inhibitory
activity are underway and the results will be reported in due
course.
All synthesized compounds were evaluated in vitro for their
ability to inhibit the catalytic activity of PARG, and for comparison
purposes, ADP-HPD was used as a positive control. PARG was iso-
lated from rat testes and purified.34 Its complementary DNA
(cDNA) was cloned. Partial cDNA clones were joined to obtain a full
length PARG DNAs and named recombinant protein G (RPG). The
plasmids were transformed into Escherichia coli. The recombinant
rat PARG protein was expressed as a glutathione S-transferase
(GST) fusion protein, GST-RPG (136 kDa), and this was employed
in the PARG activity assay35 following the method described previ-
ously.29,34 The [32P]poly(ADP-ribose) used in the assay were pre-
pared according to the method of Shimokawa et al.36 The assay
results are tabulated in Table 1. Certain of the compounds showed
anti-PARG IC50 values in the low micromolar range. More specifi-
Acknowledgments
This study was supported by a Grant-in-Aid for Cancer Research
from the Ministry of Health and Welfare of Japan (11104962). We
thank Prof. Tomohisa Nagamatsu, Department of Medical Technol-
ogy, Kumamoto Health Science University, Japan; Miss. Hiromi
Harada, National Cancer Center Research Institute, Japan, and Dr.
Yoshinobu Ishikawa, School of Pharmaceutical Sciences, University
of Shizuoka, Japan for their assistances.
cally, compound 3d (IC50= 1.0
inhibitor. Compounds 3e, 5d, 5e, 8a, 8b and 8c were also potent
inhibitors with IC50 values of 2.1, 3.1, 3.2, 3.1, 2.8 and 1.6 M,
lM) was the most active PARG
l
respectively. It is noteworthy that terephthalic acid dihydrazide
(having a 1,4 orientation about the central benzene ring) based
trihydroxyphenyl regioisomers 3d–f (IC50 values of 1.0, 2.1 and
Supplementary data
9.9 lM, respectively) were found to be more efficacious than the
isophthalic acid dihydrazide (having a 1,3 orientation about the
central benzene ring) based trihydroxyphenyl regioisomers 5d–f
(IC50 values of 3.1, 3.2 and 18.8 lM, respectively). Dihydroxy-
phenyl derivatives were less potent as compared to their respec-
tive trihydroxyphenyl derivatives. The 2,3-, 2,4- and 3,
4-dihydroxy and 2,4,6-trihydroxyphenyl derivatives analogous to
8a–h were also prepared and evaluated. However, these com-
pounds have not been reported, since they did not exhibit potent
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
06.065. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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In summary, we have designed and synthesized new phenolic
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micromolar range. Molecular docking of the most potent com-
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Compound 5e: Off-white solid; yield: 89%; mp: >300 °C; 1H NMR (300 MHz,
DMSO-d6): d 6.70 (br s, 4H), 7.67 (s, 1H), 8.07 (s, 2H), 8.19 (s, 2H), 8.40 (s, 1H),
8.62 (br s, 2H), 9.15 (br s, 4H), 11.72 (br s, 2H); TOF MS: calcd for C22H19N4O8
[M+H]+ 467.12, found 467.20. Compound 5f: brown solid; yield: 86%; mp:
>300 °C; 1H NMR (300 MHz, DMSO-d6): d 5.84 (s, 4H), 7.67 (t, J = 7.7 Hz, 1H),
8.09 (d, J = 7.7 Hz, 2H), 8.48 (s, 1H), 8.83 (s, 2H), 9.83 (s, 2H), 11.09 (br s, 4H),
12.05 (s, 2H); TOF MS: calcd for C22H19N4O8 [M+H]+ 467.12, found 467.18.
Compound 8a: yellow solid; yield: 88%; mp: >300 °C; d 6.39 (d, J = 8.4 Hz, 2H),
6.77 (d, J = 8.4 Hz, 2H), 8.53 (br s, 2H), 8.62 (s, 2H), 9.59 (br s, 2H), 11.27 (br s,
2H), 12.53 (br s, 2H); TOF MS: calcd for C16H15N4O8 [M+H]+ 391.09, found
391.30. Compound 8b: Yellow solid; yield: 86%; mp: >300 °C; 1H NMR
(300 MHz, DMSO-d6): d 6.67 (s, 4H), 8.31 (s, 2H), 8.64 (s, 2H), 9.15 (s, 4H), 11.98
(s, 2H); TOF MS: calcd for C16H15N4O8 [M+H]+ 391.09, found 391.28. Compound
8c: Brown solid; yield: 85%; mp: 239–240 °C (decomp.); 1H NMR (300 MHz,
DMSO-d6): d 3.27 (s, 1H), 3.55 (s, 1H), 6.25 (d, J = 8.4 Hz, 1H), 6.37 (d, J = 8.4 Hz,
1H), 6.78 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 8.13 (s, 1H), 8.23 (s, 1H),
8.48 (br s, 2H), 9.44 (br s, 3H), 11.25 (br s, 2H), 11.69 (br s, 1H); TOF MS: calcd
for C17H17N4O8 [M+H]+ 405.10, found 405.30. Compound 8d: brown solid;
yield: 83%; mp: 201–202 °C; 1H NMR (300 MHz, DMSO-d6): d 3.55 (s, 1H), 3.87
(s, 1H), 6.54–6.64 (m, 4H), 7.72 (s, 1H), 7.89 (s, 1H), 8.89 (br s, 6H), 11.22 (br s,
2H); TOF MS: calcd for C17H17N4O8 [M+H]+ 405.10, found 405.25. Compound
8e: Pale yellow solid; yield: 89%; mp: 283–284 °C (decomp.); 1H NMR
31. Falsig, J.; Christiansen, S. H.; Feuerhahn, S.; Bürkle, A.; Oei, S. L.; Keil, C.; Leist,
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33. General procedure for the synthesis of compounds 3a–f, 5a–f and 8a–h: A mixture
of an appropriate dihydrazide (1 mmol), an appropriate hydroxybenzaldehyde
(2 mmol) and acetic acid (2/3 drops) in methanol (25 ml) was heated under
reflux with stirring for 6–8 h. Then the reaction mixture was cooled with
stirring. Precipitated solid was collected by filtration, and washed with little
ethanol and hexane to give the target products 3a–f, 5a–f and 8a–h.
Compound 3a: Off-white solid; yield: 92%; mp: >300 °C; 1H NMR (300 MHz,
DMSO-d6): d 6.75 (t, J = 7.8 Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H), 6.99 (d, J = 7.7 Hz,
2H), 8.09 (s, 4H), 8.64 (s, 2H), 9.25 (s, 2H), 11.04 (s, 2H), 12.24 (s, 2H); TOF MS:
calcd for C22H19N4O6 [M+H]+ 435.13, found 435.14. Compound 3b: Yellow
solid; yield: 86%; mp: >300 °C; 1H NMR (300 MHz, DMSO-d6): d 6.33–6.38 (m,
4H), 7.33 (d, J = 8.4 Hz, 2H), 8.05 (s, 4H), 8.53 (s, 2H), 9.96 (br s, 2H), 11.40 (br s,
2H), 12.02 (br s, 2H); TOF MS: calcd for C22H19N4O6 [M+H]+ 435.13, found
435.15. Compound 3c: Yellow solid; yield: 89%; mp: >300 °C; 1H NMR
(300 MHz, DMSO-d6): d 6.79 (d, J = 8.1 Hz, 2H), 6.94 (d, J = 8.1 Hz, 2H), 7.26
(s, 2H), 8.02 (s, 4H), 8.28 (s, 2H), 9.34 (br s, 4H), 11.72 (br s, 2H); TOF MS: calcd
for C22H19N4O6 [M+H]+ 435.13, found 435.25. Compound 3d: Off-white solid;
yield: 95%; mp: >300 °C; 1H NMR (300 MHz, DMSO-d6): d 6.40 (d, J = 8.4 Hz,
2H), 6.80 (d, J = 8.4 Hz, 2H), 8.06 (s, 4H), 8.50 (s, 4H), 9.50 (s, 2H), 11.44 (s, 2H),
12.08 (s, 2H); TOF MS: calcd for C22H19N4O8 [M+H]+ 467.12, found 467.23.
Compound 3e: Yellow solid; yield: 88%; mp: >300 °C; 1H NMR (300 MHz,
DMSO-d6): d 6.71 (s, 4H), 8.01 (s, 4H), 8.19 (s, 2H), 8.66 (br s, 2H), 9.13 (br s,
4H), 11.69 (br s, 2H); TOF MS: calcd for C22H19N4O8 [M+H]+ 467.12, found
467.22. Compound 3f: Orange solid; yield: 83%; mp: >300 °C; 1H NMR
(300 MHz, DMSO-d6): d 5.85 (s, 4H), 8.04 (s, 4H), 8.82 (s, 2H), 9.86 (s, 2H),
11.09 (s, 4H), 12.01 (s, 2H); TOF MS: calcd for C22H19N4O8 [M+H]+ 467.12,
found 467.20. Compound 5a: Off-white solid; yield: 91%; mp: >300 °C; 1H NMR
(300 MHz, DMSO-d6): d 6.75 (t, J = 7.8 Hz, 2H), 6.87 (d, J = 7.7 Hz, 2H), 6.99 (d,
J = 7.7 Hz, 2H), 7.73 (t, J = 7.7 Hz, 1H), 8.16 (d, J = 7.7 Hz, 2H), 8.53 (s, 1H), 8.64
(s, 2H), 9.25 (s, 2H), 11.07 (s, 2H), 12.28 (s, 2H); TOF MS: calcd for C22H19N4O6
[M+H]+ 435.13, found 435.22. Compound 5b: Off-white solid; yield: 87%; mp:
>300 °C; 1H NMR (300 MHz, DMSO-d6): d 6.33–6.39 (m, 4H), 7.33 (d, J = 8.4 Hz,
2H), 7.69 (t, J = 7.7 Hz, 1H), 8.11 (d, J = 7.7 Hz, 2H), 8.47 (s, 1H), 8.54 (s, 2H),
9.98 (s, 2H), 11.42 (s, 2H), 12.08 (s, 2H); TOF MS: calcd for C22H19N4O6 [M+H]+
435.13, found 435.24. Compound 5c: Off-white solid; yield: 90%; mp: >300 °C;
1H NMR (300 MHz, DMSO-d6): d 6.79 (d, J = 7.9 Hz, 2H), 6.94 (d, J = 7.9 Hz, 2H),
7.26 (s, 2H), 7.65 (t, J = 7.7 Hz, 1H), 8.07 (d, J = 7.7 Hz, 2H), 8.28 (s, 2H), 8.42 (s,
1H), 9.35 (br s, 4H), 11.74 (br s, 2H); TOF MS: calcd for C22H19N4O6 [M+H]+
435.13, found 435.24. Compound 5d: off-white solid; yield: 93%; mp: >300 °C;
1H NMR (300 MHz, DMSO-d6): d 6.39 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H),
7.70 (t, J = 7.8 Hz, 1H), 8.13 (d, J = 7.5 Hz, 2H), 8.50 (s, 5H), 9.47 (s, 2H), 11.47 (s,
2H), 12.11 (s, 2H); TOF MS: calcd for C22H19N4O8 [M+H]+ 467.12, found 467.23.
(300 MHz, DMSO-d6):
d 2.44 (br s, 4H), 6.31 (d, J = 8.4 Hz, 2H), 6.69 (d,
J = 8.4 Hz, 2H), 8.12 (s, 2H), 8.39 (s, 2H), 9.36 (s, 2H), 11.31 (s, 2H), 11.49 (s, 2H);
TOF MS: calcd for C18H19N4O8 [M+H]+ 419.12, found 419.31. Compound 8f:
Pale yellow solid; yield: 84%; mp: 243–244 °C; 1H NMR (300 MHz, DMSO-d6): d
2.88 (br s, 4H), 6.61 (s, 4H), 7.72 (s, 1H), 7.86 (s, 1H), 8.49 (br s, 2H), 9.07 (br s,
4H), 10.97 (s, 1H), 11.13 (s, 1H); TOF MS: calcd for C18H19N4O8 [M+H]+ 419.12,
found 419.33. Compound 8g: Pale yellow solid; yield: 85%; mp: 262–263 °C;
1H NMR (300 MHz, DMSO-d6): d 1.88 (br s, 2H), 2.21–2.27 (m, 2H), 2.64 (br s,
2H), 6.35 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 8.15 (s, 2H), 8.43 (br s, 2H),
9.40 (br s, 2H), 11.46 (br s, 4H); TOF MS: calcd for C19H21N4O8 [M+H]+ 433.14,
found 433.27. Compound 8h: Pale yellow solid; yield: 81%; mp: 174–175 °C;
1H NMR (300 MHz, DMSO-d6): d 1.85 (br s, 2H), 2.16–2.22 (m, 2H), 2.61–2.66
(m, 2H), 6.57 (s, 2H), 6.61 (s, 2H), 7.69 (s, 1H), 7.85 (s, 1H), 8.50 (br s, 2H), 9.06
(br s, 4H), 10.90 (s, 1H), 11.06 (s, 1H); TOF MS: calcd for C19H21N4O8 [M+H]+
433.14, found 433.16.
34. Shimokawa, T.; Masutani, M.; Nagasawa, S.; Nozaki, T.; Ikota, N.; Aoki, Y.;
Nakagama, H.; Sugimura, T. J. Biochem. 1999, 126, 748.
35. The sample solutions in DMSO were added to 4
20 mM potassium phosphate buffer (pH 7.5), 50 mL KCl and 10 mM 2-
mercaptoethanol. The reaction was initiated by the addition of 20 L of GST-
l
M [32P]poly(ADP-ribose) in
l
PARG (15 ng), and the reaction mixture was incubated at 37 °C for 30 min. The
reaction was terminated by the addition of SDS to be final concentration of 1%.
Next, 2 lL of the reaction mixture was spotted on a poly(ethyleneimine)
cellulose TLC plate (Macherey-Nagel Co.), and the plate was developed with
0.1 M LiCl, 3 M AcOH and 3 M urea. Then, the radioactivity of the spots was
analyzed with photosensitive Fuji imaging plate BAS-2500 (Fuji Film). The
proportion of degradation products to the whole was measured, and PARG
activity was calculated.
36. Shimokawa, T.; Ogino, H.; Maeda, D.; Nakagama, H.; Sugimura, T.; Masutani, M.
Org. Chem. Insights 2009, 2, 1.