Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase β

Article abstract of DOI:10.1016/j.bmc.2012.11.024

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1-9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1- naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1- naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol β, which is a DNA repair-related pol. Compounds 3-6 strongly inhibited the growth of human colon carcinoma HCT116 p53^+/+ cells. The influence of compounds 1-9 on HCT116 p53 ^-/- cell growth was similar to that observed for HCT116 p53 ^+/+ cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol β and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol β activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53^-/- cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol β inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.

Full text of DOI:10.1016/j.bmc.2012.11.024

Bioorganic & Medicinal Chemistry 21 (2013) 403–411
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory effect of novel somatostatin peptide analogues on human cancer
cell growth based on the selective inhibition of DNA polymerase b
Isoko Kuriyama a, Anna Miyazaki b,c, , Yuko Tsuda b,c, Hiromi Yoshida a, Yoshiyuki Mizushina a,c,
⇑
⇑
a Laboratory of Food & Nutritional Sciences, Faculty of Nutrition, Kobe Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
b Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan
c Cooperative Research Center of Life Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study was designed to investigate the anticancer activity of novel nine small peptides (com-
pounds 1–9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of
mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, com-
pounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5
(Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no
carboxyl groups, selectively inhibited the activity of rat pol b, which is a DNA repair-related pol. Com-
pounds 3–6 strongly inhibited the growth of human colon carcinoma HCT116 p53+/+ cells. The influence
of compounds 1–9 on HCT116 p53ꢀ/ꢀ cell growth was similar to that observed for HCT116 p53+/+ cells.
These results suggest that the cancer cell growth suppression induced by these compounds might be
related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol b and cancer cell growth
among the nine compounds tested. This compound specifically inhibited rat pol b activity, but had no
effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sul-
fonate (MMS) treatment synergistically suppressed HCT116 p53ꢀ/ꢀ cell growth compared with MMS
alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results,
the influence of compound 4, a specific pol b inhibitor, on the relationship between DNA repair and can-
cer cell growth is discussed.
Received 30 October 2012
Revised 12 November 2012
Accepted 16 November 2012
Available online 29 November 2012
Keywords:
Peptides
DNA polymerase b
Enzyme inhibitor
Methyl methane sulfonate (MMS)
DNA repair
Anticancer
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
other bioactive hormones including glucagons, insulin, gastrin
and secretin, and also affects the regulation of cell prolifera-
DNA polymerase (pol, DNA-dependent DNA polymerase,
E.C.2.7.7.7) catalyses the addition of deoxyribonucleotides to the
3’-hydroxyl terminus of primed double-stranded DNA (dsDNA)
molecules.1 The human genome encodes 15 pols that conduct cel-
lular DNA synthesis.2 Eukaryotic cells reportedly contain the fol-
tion.7–9 These diverse pharmacological actions are dependent on
their affinities to somatostatin receptors 1–5 (SSTRs1-5), which
are widely distributed not only on human tissues including the
brain, gastrointestinal tract, and pancreas but also on several can-
cer cells.10,11 Somatostatin analogues are used as diagnostic
agents and drugs to treat endocrine tumors,12–15 but their clinical
use as antitumor agents is restricted because of their anti-secre-
tory effects and poor oral bioavailability. To clarify the functions
of their respective SSTRs and develop more potent analogues,
many efforts have been undertaken to identify SSTRs subtype
selective analogues. Kéri et al. identified the somatostatin struc-
lowing three replicative types: (i) pols
mitochondrial pol , and (iii) thirteen repair types, namely pols b,
d, , f, , h, , k, and , REV1 and terminal deoxynucleotidyl
a, d and e, (ii)
c
e
g
i
,
j
l
m
transferase (TdT).3 DNA metabolic enzymes such as pols are not
only essential for DNA replication, repair and recombination, but
also are involved in cell division. Selective inhibitors of these en-
zymes are considered to be potentially useful anticancer and anti-
parasitic agents because they have been shown to suppress human
cancer cell proliferation and are cytotoxic.4–6
tural derivative TT-232, H-D-Phe-c(Cys-Tyr-D-Trp-Lys-Cys)-Thr-
NH2, which exhibits strong antitumor activity in vitro and
in vivo without other SRIF effects, including anti-secretory ac-
tion.16,17 Its antitumor activity was not exhibited through interac-
tions with SSTRs although it exhibited a small amount of binding
affinity to SSTRs. Previously, we studied the structure-activity
Somatostatin-14 (SRIF) is a natural cyclic tetradecapeptide
which inhibits the release of growth hormones, suppresses many
relationships of derivatives of Tyr-D-Trp-Lys, the active sequence
⇑
Corresponding authors. Tel.: +81 78 974 1551x3232; fax: +81 78 974 5689
(Y.M.).
in TT-232, and identified some compounds with very potent anti-
proliferative activity toward several cancer cells.18 Unlike the cyc-
E-mail addresses: miyazaki@pharm.kobegakuin.ac.jp (A. Miyazaki), mizushin@-
nutr.kobegakuin.ac.jp (Y. Mizushina).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.024

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I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
lic compounds reported as general somatostatin analogues, these
compounds are small peptide mimetics with features of linear
structure and consisting of only two amino acids and one hydro-
phobic group. Furthermore, the small linear compound tert-butyl-
oxycarbonyl (Boc)-Tyr-
pols ( , b, , k) inhibition, but then showed almost no cancer cell
growth inhibitory activity.19 In this study, we prepared the new
D-Trp-1-naphthylamide exhibited potent
a
j
small linear compounds 1–9 based on the Boc-Tyr-D-Trp-1-naph-
O
X
Y
N
H
O
Boc-[X]-[Y]-1-naphthylamide
Compound
X
Y
Compound
X
Y
O
O
O
H
N
H
N
Tyr
Tyr
1
5
O
OH
Asp
Leu
O
H
N
H
N
2
Tyr
6
Tyr
O
OH
Glu
Val
O
O
2
H
N
H
N
7
8
9
3
Tyr
O
4
H
N
Phe
O
H
N
4
O
6
Tyr
H
N
Ile
O
H
Tyr=
N
OH
Figure 1. Structures of the synthesized novel peptide analogues of somatostatin. Compound 1, Boc-Tyr-Asp-1-naphthylamide; compound 2, Boc-Tyr-Glu-1-naphthylamide;
compound 3, Boc-Tyr-Phe-1-naphthylamide; compound 4, Boc-Tyr-Ile-1-naphthylamide; compound 5, Boc-Tyr-Leu-1-naphthylamide; compound 6, Boc-Tyr-Val-1-
naphthylamide; compound 7, Boc-NH-(CH2)2-1-naphthylamide; compound 8, Boc-NH-(CH2)4-1-naphthylamide and compound 9, Boc-NH-(CH2)6-1-naphthylamide.

I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
405
thylamide sequence and studied their structure-activity relation-
2. Results
ships. Pols are considered a promising target for anticancer drugs.
In particular, pol b plays an important role in base excision repair
(BER) of DNA bases damaged by many anticancer drugs and exog-
enous alkylating agents.20–22 Pol b is the smallest human pol and
the first discovered adaptive polymerase. By X-ray crystallogra-
phy, it was demonstrated that the full-length pol b protein
(39 kDa) was divided into two structural domains, an 8 kDa N-
terminal and a 31 kDa C-terminal domain.23 It is reported that
DNA and other pol b inhibitory compounds first bind to the
2.1. Effect of somatostatin peptide analogues on mammalian
pol activity
The structures of novel nine chemically synthesized somato-
statin peptide analogues (compounds 1–9) are shown in Figure 1.
The inhibitory activity of each compound at 200
gated using calf pol , rat pol b and human pol . The mammalian
pols , b and were chosen as a representative replicative pol (B-
lM was investi-
a
j
a
j
8 kDa domain binding site,24,25 which is formed by 4
a-helices,
family pols), a repair/recombination-related pol (X-family pols)
packed as two antiparallel pairs.26,27 Milon et al. performed dock-
ing studies of pol b and pamoic acid, a compound shown to inhi-
bit deoxyribose phosphate lyase and pol b, and found that it
bound in a single pocket at the surface of the 8 kDa domain. This
pocket contained a positive groove and hydrophobic area where
the DNA was also bound.28 This result promoted us to design no-
vel compounds 1–9. Compounds 1–6 contained more hydropho-
and a translation synthesis (TLS) repair pol (Y-family pols), respec-
tively.2,3 As shown in Figure 2, the activities of pols
a and j were
not affected (the pol relative activity was >50%). Compounds 3–6,
which contain tyrosine (Tyr) and a hydrophobic amino acid (no
carboxyl group), inhibited the activity of pol b. Compound 4
showed the strongest pol b inhibition followed in order by com-
pound 3 > compound 5 > compound 6.
bic and negatively charged amino acids instead of the
residue found in the parent compound, and compounds 7–9 con-
tained only an aliphatic amino acid and a 1-naphthylamide group
in order to confirm the necessity of the Tyr-D-Trp sequence.
D-Trp
When activated DNA (bovine deoxyribonuclease I-treated DNA)
was used as the DNA template-primer substrate instead of synthe-
sized DNA [poly(dA)/oligo(dT)18 (A/T = 2/1)], and dNTP was used as
the nucleotide substrate instead of dTTP, the inhibitory effects of
these compounds did not change (data not shown).
The biochemical action of the novel somatostatin peptide ana-
logues 1–9, including their in vitro inhibition of mammalian pol
species, on DNA replication and repair of human colon carcinoma
(HCT116) cells treated with a DNA damaging agent, methyl meth-
ane sulfonate (MMS), were studied. The potential anticancer activ-
ities of the peptide somatostatin analogues based on their DNA
repair-related pol inhibition are discussed. The structures of novel
compounds 1–9, all peptide mimetics, make them unique pol
inhibitors as other published inhibitors generally contain a hydro-
xyl group, carbonyl group and aromatic heterocycle.
2.2. Inhibitory effect of compound 4 on eleven mammalian pol
species
The activity of compound 4 toward eleven mammalian pols: pol
c
(from the A-family), pols
(X-family), and pols and
Compound 4 dose-dependently inhibited the activity of rat pol b,
with an IC50 value of 11.5 M (Table 1). In contrast, compound 4
a
, d and
j
e (B-family), pols b, k, l and TdT
g
,
i
(Y-family) was then investigated.2,3
l
Calf pol α
Rat pol β
Human pol κ
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
0
20
40
60
80
100
Pol relative activity (%)
Figure 2. Effect of novel somatostatin peptide analogues (compounds 1–9) on the activity of mammalian pols. Each compound at 200
pol b and human pol (0.05 units each). Pol activity was measured as described in the Experimental procedures, and is shown as a percentage of enzyme activity in the
absence of the compound, which was taken as 100%. Data are the mean S.D. of three independent experiments.
lM was incubated with calf pol a, rat
j

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I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
Table 1
Inhibition of mammalian pol activities by compound 4
hydrophobic amino acid (no carboxyl group), thus the tyrosine
group must play a key role in the activities of these compounds.
On the other hand, compounds 1, 2 and 7–9 hardly inhibited hu-
man cancer cell growth. In terms of the growth inhibitory effect,
the ranking was compound 4 > compound 3 > compound 6 > com-
pound 5. The influence of compounds 1–9 on HCT116 p53ꢀ/ꢀ cell
growth showed the same pattern as that observed for HCT116
p53+/+ cells (Fig. 3B), suggesting that p53 protein expression had
no effect on the cell growth suppression induced by somatostatin
peptide analogues. These results suggest that the inhibition of can-
cer cell growth by these compounds may be related to the inhibi-
tion of pol activity, in particular, the inhibition of pol b by
compound 4 is likely important for HCT116 cell proliferation. Since
compound 4 did not influence the cell growth of a human normal
cell line, human dermal fibroblasts (HDF) from the skin (data not
shown), this compound might be selective cytotoxic effect against
cancer cells. Therefore, additional experiments were carried out on
compound 4 in the latter part of this study.
Mammalian pols
IC50 values (lM)
[A-Family]
Human pol
c
>200
[B-Family]
Calf pol
Human pol d
Human pol
a
>200
>200
>200
e
[X-Family]
Rat pol b
Human pol k
11.5 0.7
>200
Human pol
Calf TdT
l
>200
>200
[Y-Family]
Human pol
Mouse pol
Human pol
g
i
j
>200
>200
>200
Compound 4 was incubated with each enzyme (0.05 units); pol
activity in the absence of inhibitor was set to 100%; data are the
mean S.D. (n = 3)
2.4. Reproductive cell death in p53+/+ and p53ꢀ/ꢀ cells
Pol b and p53 play a significant role in base excision repair
(BER).29 Therefore, to determine whether compound 4-induced cell
growth suppression is the cause of the inhibition of pol b, the ef-
fects of compound 4 on BER-induced by DNA alkylation damage
in HCT116 p53+/+ or p53ꢀ/ꢀ cells were investigated using a clono-
genic assay. Alkylation damage induced by MMS is repaired exclu-
sively by the BER pathway.30 HCT116 cells with or without p53
were treated with 1 mM MMS for 1 h followed by 1% DMSO (vehi-
exhibited no effect toward the other 10 mammalian pols. In partic-
ular, it was interesting that compound 4 did not affect the activities
of pol k,
l and TdT which are thought to share similar homology
and three-dimensional structures with pol b, although these pols
belong to the X-family.
To ascertain whether the inhibition induced by compound 4 re-
sulted from its ability to bind to DNA or pol b protein, the thermal
transition of DNA in the presence or absence of the compound was
measured. The interaction of compound 4 with dsDNA was inves-
tigated by measuring the melting temperature (Tm) of dsDNA in
the presence of excess compound using a spectrophotometer
equipped with a thermoelectric cell holder. An alteration in ther-
mal transition (i.e., Tm) was not observed within the compound
concentration range tested, whereas a typical intercalating com-
cle control) or compound 4 at 13.2 lM or 13.7 lM based on the
LD50 values of p53+/+ or p53ꢀ/ꢀ, respectively, for 24 h (Fig. 4). The
surviving fraction obtained after 1 mM MMS treatment was
0.05 0.01 and 0.63 0.13 for HCT116 p53+/+ and HCT116 p53ꢀ/
ꢀ, respectively, suggesting that HCT116 p53+/+ cells could repair
the DNA damaged by MMS through a p53 dependent BER pathway,
but the HCT116 p53ꢀ/ꢀ cells could not. When exposed to com-
pound 4, cell death was slightly favored for HCT116 cells with or
without p53. Upon treatment with MMS and compound 4, p53
wild type cells showed an approximately two fold reduction in clo-
nogenic survival compared with the vehicle control (Fig. 4A). Fur-
thermore, compared to the vehicle control of p53-deficient cells,
treatment with compound 4 resulted in a significant reduction
(25-fold) in clonogenic survival (Fig. 4B). These data suggested that
compound 4 induced cell growth suppression by the inhibition of
pol b.
pound used as a positive control (ethidium bromide, 15
lM) pro-
duced clear change in the thermal transition. We also
a
investigated if excess amounts of nucleic acid [poly(rC)] or protein
[bovine serum albumin (BSA)] prevented the inhibitory effects of
compound 4 in order to determine whether the observed inhibi-
tory effect resulted from non-specific adhesion of these potentially
interfering molecules to pol b or from selective binding to specific
sites. Poly(rC) and BSA had little or no influence on the inhibitory
effects of compound 4 on pol b suggesting that compound 4 bound
selectively to the target enzyme molecules. These observations
suggested that compound 4 did not act as a DNA intercalating
agent but that it bound directly to the enzyme and selectively
inhibited its activity.
2.5. Effect of compound 4 on apoptotic cell death
To examine whether compound 4 induced apoptosis upon
treatment, DNA fragmentation was analyzed by electrophoresis
(Fig. 5A). When HCT116 p53+/+ cells and HCT116 p53ꢀ/ꢀ cells were
Taken together, these results suggested that compound 4 was a
potent and selective inhibitor of mammalian pol b.
treated with compound 4 at 13.2 lM or 13.7 lM, respectively, for
2.3. Effects of somatostatin peptide analogues on cultured
human cancer cell growth
24 h, these cells clearly underwent DNA fragmentation (lanes 2
and 4 in Fig. 5A). When HCT1116 cells with or without p53 were
exposed to 1 mM MMS for 1 h, the observed DNA fragmentation
was moderate (lanes 5 and 6 in Fig. 5A). These results suggested
that compound 4 and MMS induced apoptosis in both HCT116
p53+/+ cells and HCT116 p53ꢀ/ꢀ cells.
Figure 5B shows the extent of apoptotic cells by terminal deoxy-
nucleotidyl transferase dUTP nick end labeling (TUNEL) analysis.
The percentage of HCT116 p53+/+ apoptotic cells after treatment
with compound 4 was three fold higher than that of HCT116
p53ꢀ/ꢀ cells, suggesting that apoptotic cell death might be induced
by a p53 dependent-pathway. MMS also induced apoptosis in
HCT116 p53+/+ cells, but hardly influenced HCT116 p53ꢀ/ꢀ cells
compared with the control as shown by the high clonogenic sur-
Pols have recently emerged as important cellular targets for
chemical intervention in the development of anticancer agents.
The somatostatin peptide analogues (compounds 1–9) could there-
fore prove useful in chemotherapy. We investigated the cytotoxic
effects of the nine compounds against two HCT116 human colon
carcinoma cultured cell lines, one wild-type p53 (p53+/+) and one
deletion mutant of p53 (p53ꢀ/ꢀ). As shown in Figure 3A, 200
lM
of compounds 3–6 strongly inhibited the growth of HCT116
p53+/+ cells. Treatment with compounds 4 and 3 resulted in the
strongest cell growth suppression with LD50 values of 13.2 and
30.7 lM, respectively. These compounds contain a tyrosine and a

I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
407
20 μM
A
B
200 μM
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
0
20
40
60
80
100
0
20
40
60
80
100
Rate of HCT116 p53+/+ cell growth (%)
Rate of HCT116 p53-/- cell growth (%)
Figure 3. Effect of novel somatostatin peptide analogues (compounds 1–9) on the proliferation of HCT116 human colon carcinoma cultured cell growth. Each compound at
20 lM (gray bar) and 200 l
M (black bar) was added to a culture of HCT116 cells with wild-type p53 (HCT116 p53+/+) (A) and their isogenic derivatives that lack p53 (HCT116
p53ꢀ/ꢀ) (B). The cells were incubated for 24 h, and the rate of cell growth inhibition was determined by WST-1 assay. Cell growth in the absence of the compound was taken
as 100%. Data are the mean S.D. of five independent experiments.
vival rate of HCT116 p53ꢀ/ꢀ cells in the presence of MMS alone
(Fig. 4B). Compound 4 was five fold more potent at inducing apop-
tosis than MMS. Moreover, when MMS and compound 4 were com-
bined together, the extent of apoptotic induction was significantly
higher than treatment with either alone for both HCT116 cells with
or without p53. This data indicated that compound 4 might further
enhance MMS-induced apoptosis.
somal breakage by methylating agents such as MMS (Fig. 5). More-
over, incompletely repaired DNA damage causes chromosomal
changes and may act as a trigger of DNA damage-induced apopto-
sis.31 This phenomenon was observed in the surviving fraction of
HCT116 cells without p53 which underwent DNA damage-induced
apoptosis to approximately the same extent as HCT116 cells with
p53. Recently, interest is growing in inducing apoptosis as a new
target for cancer chemotherapy.32 Apoptosis is an important series
of events that leads to programmed cell death and is essential for
tissue development and homeostasis. Several studies have indi-
cated that anticancer drugs or cancer chemopreventive agents
act by inducing apoptosis in various cancer cells. Additionally,
the initiation of apoptosis appears to be a common mechanism
of many new chemotherapeutic anticancer agents.33
As shown in Figure 5, compound 4 induced apoptosis via a p53-
independent pathway in HCT116 cells. The sensitivity of p53 defec-
tive cells to the DNA base-damaging agent MMS34 suggested that
certain p53-deficient cancers may be preferentially sensitive to
chemotherapeutic base-damaging agents like cyclophosphamide
which trigger repair by BER. However, as has been noted by other
researchers, there have been surprisingly few studies on the role of
p53 in the cellular response to DNA-damaging chemotherapeutic
agents for the treatment of epithelial cancers that are not intrinsi-
cally prone to undergo apoptosis.35 Compound 4, therefore, should
be considered a potentially useful anticancer chemotherapeutic
agent.
3. Discussion
We examined the effect of novel nine somatostatin peptide ana-
logues (Fig. 1) on the activities of mammalian pols and human co-
lon carcinoma HCT116 cells with or without p53. Boc-Tyr-Phe-1-
naphthylamide (compound 3) and Boc-Tyr-Ile-1-naphthylamide
(compound 4) potently inhibited the activities of mammalian pols
and suppressed human cancer cell growth (Figs. 2 and 3). In partic-
ular, compound 4 was a selective inhibitor of pol b (Table 1). Inter-
estingly, these compounds were derived from the parent
compound by substituting
D-Trp with a more hydrophobic and
smaller amino acid. The mechanisms of pol b specific inhibition
by compound 4 among the 11 mammalian pols tested remain un-
clear and will require further study. The suppression of cell growth
showed the same trend as the inhibition of pol b among the
somatostatin peptide analogues suggesting that the suppression
of cell growth is related to the activity of pol b. To determine
whether compound 4-induced cell growth suppression is the result
of the inhibition of pol b, the effect of this compound on BER-in-
duced DNA alkylation damage in HCT116 p53+/+ or p53ꢀ/ꢀ cells
was investigated using a clonogenic assay. Wild-type p53 protein
can significantly stimulate BER in vitro. Furthermore, p53 stabilizes
the interaction between pol b and abasic DNA. Hence, after 1 mM
MMS treatment, cell survival was 12.6-fold higher for HCT116
p53ꢀ/ꢀ cells than for HCT116 p53+/+ cells (Fig. 4). Cells lacking
pol b are highly sensitive to induction of apoptosis and chromo-
4. Conclusion
The novel somatostatin peptide analogues, especially com-
pound 4, selectively inhibited the activity of mammalian pol b
among the 11 mammalian pol species tested (Fig. 2 and Table 1).
The hydrophobic character of the pol b binding site is a very impor-
tant determinant of its ability to interact with inhibitors and DNA.

408
I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
Figure 4. Clonogenic survival of HCT116 cells after treatment with compound 4
and MMS. (A) Treatment schedules combining 1 mM MMS for 1 h and 13.2
compound 4 for 24 h of HCT116 p53+/+ cells. (B) Treatment schedules combining
1 mM MMS for 1 h and 13.7
M compound 4 for 24 h of HCT116 p53ꢀ/ꢀ cells. Cells
were treated with (closed circle) or without (open circle) compound 4. Data are the
mean S.D. of three independent experiments.
lM
Figure 5. Apoptotic effect of compound 4 or MMS on HCT116 cells. (A) DNA
fragmentation of compound 4 or MMS treated cells by agarose gel electrophoresis.
M, marker; lane 1, untreated control of HCT116 p53+/+ cells; lane 2, HCT116 p53+/+
l
cells were treated with 13.2
lM compound 4 for 24 h; lane 3, untreated control of
HCT116 p53ꢀ/ꢀ cells; lane 4, HCT116 p53ꢀ/ꢀ cells were treated with 13.7
l
M
compound 4 for 24 h; lane 5, HCT116 p53+/+ cells were treated with 1 mM MMS for
1 h; lane 6, HCT116 p53ꢀ/ꢀ cells were treated with 1 mM MMS for 1 h. Total DNA
was then extracted and analyzed by 1.5% agarose gel electrophoresis. A photograph
of the ethidium bromide-stained gel is shown. (B) Apoptotic induction by
compound 4 and/or MMS. HCT116 p53+/+ cells (gray bar) and HCT116 p53ꢀ/ꢀ cells
An inhibitor containing a relatively small side chain may be favor-
able for interaction with the binding pocket. Compounds 1 and 2
contained Asp and Glu, respectively, but showed almost no activ-
ity. Other structural features, including the number of carboxyl
groups, must play a role in achieving the proper orientation for
binding in the pocket, although further study is required to clarify
the structure-activity relationship of candidate molecules. Com-
pound 4 potently suppressed human colon carcinoma HCT116 cell
growth with or without p53 (Fig. 3). Treatment of HCT116 p53ꢀ/ꢀ
cells with compound 4 significantly enhanced cell multiplication
suppression upon pre-addition of MMS (Fig. 4). Therefore, com-
pound 4 has great potential for development as an anticancer
agent and could be an effective clinical anticancer chemotherapeu-
tic in combination with DNA damaging agents, such as MMS.
(black bar) were incubated for 24 h with 13.2 or 13.7 lM compound 4, respectively,
for 1 h with 1 mM MMS, and for 1 h with 1 mM MMS followed by compound 4 for
24 h (1 mM MMS + compound 4). Cells were detected by TUNEL assay using an
ApopTag Red In Situ Apoptosis Detection Kit. Apoptotic cells were individually
counted from a total of at least 200 cells (for each condition). Values are shown as
the mean S.D. for three independent experiments.
midine 50-triphosphate (dTTP) (43 Ci/mmol), was obtained from
MP Biomedicals LLC (Solon, OH, USA). All other reagents were of
analytical grade and were purchased from Nacalai Tesque Inc.
(Kyoto, Japan). 1-Naphthylamine was purchased from Tokyo
Chemical Industry Co., Ltd (Tokyo, Japan). Amino acids and other
reagents were purchased from Watanabe Chemical Industries,
Ltd (Hiroshima, Japan). Analytical and semi-preparative RP-HPLC
columns were Waters 600E with COSMOSIL 5C18-AR-II
(4.6 ꢁ 250 mm) and 5C18-AR-II (20 ꢁ 250 mm), respectively. Mo-
bile solvents used were (A) 0.05% TFA in water, (B) 0.05% in aceto-
nitrile, (C) water and (D) acetonitrile. All compounds were
analyzed by employing the following gradient program: after
maintenance (A):(B) 90:10 for 5 min, and from (A):(B) 90:10 to
5. Experimental procedure
5.1. General
The chemically synthesized DNA template, poly(dA) was pur-
chased from Sigma–Aldrich Inc. (St. Louis, MO, USA). The oli-
go(dT)18 DNA primer was customized by Sigma–Aldrich Japan
K.K. (Hokkaido, Japan). The radioactive nucleotide, [3H]-deoxythy-

I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
409
10:90 over 40 min. Mass spectra were recorded on a Bruker micro
TOF-Q with electrospray ionization (ESI-TOF-MS). 1H NMR spectra
were recorded in CDCl3 or DMSO on a Bruker DPX-400 (400 MHz).
Silica gel 60 (0.063–0.200 mm, 70–230 mesh by Merck) and Kiesel-
gel G 60 (Merck KGaA, Darmstadt, Germany) were used for open
column chromatography and thin layer chromatography,
respectively.
4.86 (br s, 1H), 4.13 (br s, 1H), 2.91 (br s, 1H), 2.67 (br s, 2H),
1.27 (s, 9H).
5.2.1.2. Boc-Tyr-Glu-1-naphthylamide (2).
The residue was
precipitated with hexane to give a white solid (45.7%), mp 117–
120 °C, Rf 0.51 (CHCl3:MeOH:H2O = 8:3:1), RT 33.7 min, ESI-MS m/
z
Calcd [M+H]+ 536.23. Found 536.27. Anal. Calcd for
C
29H33N3O7ꢂ0.9H2O: C, 63.12; H, 6.36; N, 7.62. Found: C, 63.00;
5.2. Chemistry
H, 6.26; N, 7.65. 1H NMR (CDCl3) d 10.05 (s, 1H), 9.19 (br s, 1H),
8.23 (d, J = 7.6 Hz, 1H), 8.05 (br s, 1H), 7.94 (td, J = 1.9 and 5.9 Hz,
1H), 7.79 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.57–7.48 (m,
3H), 7.06 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.3 Hz, 1H), 6.63 (d,
J = 8.4 Hz, 2H), 4.65 (q, J = 7.0 Hz, 1H), 4.16 (m, 1H), 2.91 (dd,
J = 4.0 and 14 Hz, 1H), 2.67 (dd, J = 10 and 14 Hz, 1H), 2.42–2.31
(br s, 2H), 2.14 (br s, 1H), 2.10 (br s, 1H), 1.27 (s, 9H).
Novel compounds were synthesized by a solution phase meth-
od. Starting amino acids and aliphatic amino acids with the N-Boc
group without a protecting group on the side chain were used, ex-
cept for Asp and Glu whose side chains were protected by a benzyl
ester (OBzl) group. For compounds 1–6, the corresponding amino
acid as staring material was coupled with 1-naphthylamine using
benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophos-
phate (PyBOP) in N,N-dimethylformamide (DMF) containing diiso-
propylethylamine (DIPEA). For the coupling of Boc-Asp(OBzl)-OH
and Boc-Glu(OBzl)-OH, the appropriate amino acid was coupled
with 1-naphthylamine by a mixed anhydride method. The Boc
group of the resulting compound was removed by 4 M HCl/dioxane
to produce the corresponding HCl salt, which was coupled with
Boc-Tyr-OH by use of PyBOP to give compounds 3–6. Compounds
1 and 2 were prepared as follows using a further deprotection step.
The OBzl group of compounds 1 and 2 was deprotected by catalytic
hydrogenation using Pd-Black in 50% AcOH containing a small
amount of MeOH for 2 h. For preparation of compounds 7–9, the
N-Boc forms of 3-aminopropionic acid, 5-aminovaleric acid and
7-aminoheptanoic acid were used as starting materials, and cou-
pled with 1-naphthylamine using a mixed anhydride method. Final
products were purified by RP-HPLC and found to be greater than
98% pure. Compounds 1 and 2 were purified using the following
gradient program: from (C):(D) 70:30 to (C):(D) 40:60 over
5 min, then (C):(D) 10:90 over 10 min. Compounds 3–6 were puri-
fied by employing the following gradient program: from (C):(D)
70:30 to (C):(D) 50:50 over 5 min, then (C):(D) 20:80 over
15 min. Compounds 7–9 were purified by employing the following
gradient program: from (C):(D) 70:30 to (C):(D) 40:60 over 5 min,
then (C):(D) 30:70 over 10 min.
5.2.1.3. Boc-Tyr-Phe-1-naphthylamide (3).
The residue was
precipitated with hexane and a small amount of ether to give a
white solid (60.0%), mp 207–210 °C, Rf 0.44 (CHCl3:MeOH:A-
cOH = 90:8:2), RT 40.3 min, ESI-MS m/z Calcd [M+H]+ 554.26.
Found 554.29. Anal. Calcd for C33H35N3O5ꢂ0.1H2O: C, 71.35; H,
6.39; N, 7.57. Found: C, 71.30; H, 6.37; N, 7.64. 1H NMR (CDCl3) d
10.04 (s, 1H), 9.16 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.92 (d,
J = 7.9 Hz, 1H), 7.76 (t-like, 2H), 7.54–7.45 (m, 4H), 7.37–7.21 (m,
5H), 7.00 (d, J = 8.3 Hz, 2H), 6.88 (d, J = 8.5 Hz, 1H), 6.62 (d,
J = 8.3 Hz, 2H), 4.93 (q, J = 7.4 Hz, 1H), 4.12 (m, 1H), 3.16 (dd,
J = 6.7 and 13 Hz, 1H), 3.07 (dd, J = 7.9 and 14 Hz, 1H), 2.81 (dd,
J = 4.1 and 14 Hz, 1H), 2.62 (dd, J = 10 and 13 Hz, 1H), 1.29 (s, 9H).
5.2.1.4. Boc-Tyr-Ile-1-naphthylamide (4).
The residue was
precipitated with ether to give a white solid (89.0%), mp 197–
200 °C, Rf 0.48 (CHCl3:MeOH:AcOH = 90:8:2), RT 39.7 min, ESI-MS
m/z Calcd [M+H]+ 520.27. Found 519.90. Anal. Calcd for
C
30H37N3O5ꢂ1.2H2O: C, 66.57; H, 7.34; N, 7.77. Found: C, 66.44;
H, 7.29; N, 7.76. 1H NMR (CDCl3) d 10.12 (s, 1H), 9.16 (s, 1H),
8.06 (br s, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.94 (td, J = 1.5 and 5.0 Hz,
1H), 7.79 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 7.1 Hz, 1H), 7.57–7.49 (m,
3H), 7.38 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.5 Hz, 1H), 6.63 (d,
J = 8.3 Hz, 2H), 4.60 (t, J = 8.2 Hz, 1H), 4.19 (m, 1H), 2.90 (dd,
J = 4.0 and 7.2 Hz, 1H), 2.69 (dd, J = 6.9 and 10 Hz, 1H), 1.92 (br s,
1H), 1.61 (br s, 1H), 1.32–1.27 (m, 10H), 1.02 (d, J = 6.7 Hz, 3H),
0.91 (t, J = 7.2 Hz, 3H).
5.2.1. General procedure for the synthesis of Boc-[X]-[Y]-1-
naphthylamide [X: Tyr, Y: Asp (1), Glu (2), Phe (3), Ile (4), Leu (5),
Val (6)]
5.2.1.5. Boc-Tyr-Leu-1-naphthylamide (5).
The residue was
Boc-Tyr-OH (720 mg, 2.60 mmol) and H-[Y]-1-naphthylamide
[prepared from Boc-[Y]-1-naphthylamide (1.00 g, 2.60 mmol) and
4 M HCl/dioxane (6.40 mL, 25.6 mmol)] in DMF (50 mL) were cou-
pled by PyBOP (1.47 g, 2.80 mmol) and DIPEA (2.68 mL,
15.3 mmol) at room temperature overnight. After removal of sol-
vent, the residue was extracted with AcOEt, washed with 10% citric
acid, 5% NaHCO3 and water, then dried over Na2SO4. After removal
of Na2SO4 by filtration, the filtrate was evaporated. For compounds
1 and 2, to a solution of the resulting compounds in MeOH (20 mL)
containing a drop of H2O, a small amount of Pd-black was added.
H2 gas was passed through the solution for 2 h. The catalyst was re-
moved by filtration, and the filtrate was evaporated.
precipitated with ether to give a white solid (53.4%), mp 205–
209 °C, Rf 0.37 (CHCl3:MeOH:AcOH = 90:8:2), RT 40.4 min, ESI-MS
m/z Calcd [M+H]+ 520.27. Found 520.31. Anal. Calcd for
C30H37N3O5ꢂ0.4H2O: C, 68.39; H, 7.23; N, 7.98. Found: C, 68.27;
H, 7.28; N, 8.11. 1H NMR (CDCl3) d10.03 (s, 1H), 9.16 (s, 1H), 8.16
(d, J = 7.9 Hz, 1H), 8.03 (t-like, 1H), 7.94 (td, J = 1.7 and 4.3 Hz,
1H), 7.79 (d, J = 8.1 Hz, 1H), 7.61–7.48 (m, 4H), 7.06 (d, J = 8.4 Hz,
2H), 6.92 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 4.68 (q,
J = 7.5 Hz, 1H), 4.16 (m, 1H), 2.89 (dd, J = 4.0 and 14 Hz, 1H), 2.67
(dd, J = 10 and 14 Hz, 1H), 1.78–1.68 (m, 3H), 1.31 (s, 9H), 0.99
(d, J = 6.3 Hz, 2H), 0.95 (d, J = 6.2 Hz, 2H).
5.2.1.6. Boc-Tyr-Val-1-naphthylamide (6).
The residue was
5.2.1.1. Boc-Tyr-Asp-1-naphthylamide (1).
precipitated with hexane to give a white solid (40.0%), mp 125–
The residue was
precipitated with ether to give a white solid (66.7%), mp 197–
200 °C, Rf 0.38 (CHCl3:MeOH:AcOH = 90:8:2), RT 38.1 min, ESI-MS
m/z Calcd [M+H]+ 506.26. Found 506.26. Anal. Calcd for
129 °C, Rf 0.46 (CHCl3:MeOH:H2O = 8:3:1), RT 34.0 min, ESI-MS m/
z
Calcd [M+H]+ 522.22. found 522.22. Anal. Calcd for
C29H35N3O5ꢂ0.5H2O: C, 67.69; H, 7.05; N, 8.17. Found: C, 67.85;
C
28H31N3O7ꢂ0.3CH3CNꢂ2.5MeOH: C, 60.83; H, 6.88; N, 7.53. Found:
H, 7.05; N, 8.29. 1H NMR (CDCl3) d 10.11 (s, 1H), 9.17 (s, 1H),
8.06 (br s, 1H), 7.97–7.93 (m, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.64 (d,
J = 7.1 Hz, 1H), 7.58–7.49 (m, 3H), 7.07 (d, J = 8.4 Hz, 2H), 6.98 (d,
J = 8.5 Hz, 1H), 6.64 (d, J = 8.4 Hz, 2H), 4.59 (t, J = 7.9 Hz, 1H), 4.20
(m, 1H), 2.91 (dd, J = 4.0 and 14 Hz, 1H), 2.69 (dd, J = 10 and
C, 60.85; H, 7.05; N, 7.51. 1H NMR (CDCl3) d 10.21 (s, 1H), 9.19 (br s,
1H), 8.43 (br s, 1H), 8.10 (br s, 1H), 7.93 (t, J = 4.7 Hz, 1H), 7.77 (d,
J = 8.2 Hz, 1H), 7.66 (d, J = 7.1 Hz, 1H), 7.54–7.47 (m, 3H), 7.05 (d,
J = 8.1 Hz, 2H), 6.83 (d, J = 7.9 Hz, 1H), 6.62 (d, J = 8.1 Hz, 2H),

410
I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
14 Hz, 1H), 2.21–2.12 (m, 1H), 1.32 (s, 9H), 1.04 (d, J = 6.7 Hz, 3H),
1.00 (d, J = 6.7 Hz, 3H).
sidered 100%, and the remaining activity at each concentration of
the inhibitor was determined relative to this value. One unit of
pol activity was defined as the amount of each enzyme that cata-
lyzed the incorporation of 1 nmol of dNTP (i.e., dTTP) into synthetic
DNA template-primers in 60 min at 37 °C and under normal reac-
tion conditions.30,31
5.2.2. General procedure for the synthesis of Boc-NH-(CH2)n-1-
naphthylamide [n = 2 (7), 4 (8), 6 (9)]
To a solution of Boc-NH–(CH2)2–OH (1.00 g, 4.10 mmol) in tet-
rahydrofuran (THF) (30 mL), triethylamine (TEA) (0.70 mL,
5.00 mmol) and isobutyl chroloformate (0.60 mL, 4.50 mmol) were
added and stirred at ꢀ15 °C for 15 min. To this solution, 1-naph-
thylamine (0.60 g, 4.10 mmol) in DMF (40 mL) containing TEA
(0.70 mL, 5.00 mmol) was added and stirred at room temperature
overnight. After removal of solvent, the residue was extracted with
AcOEt, washed with 10% citric acid, 5% NaHCO3 and water, then
dried over Na2SO4. After removal of Na2SO4 by filtration, the filtrate
was evaporated. The residue was precipitated with ether to give a
crystalline solid.
5.4. Cell culture and measurement of cell viability
To investigate the effects of novel somatostatin peptide ana-
logues (compounds 1–9) on cultured human cancer cells, HCT116
human colon carcinoma cells with wild-type p53 (HCT116 p53+/
+) and their isogenic derivatives lacking p53 (HCT116 p53ꢀ/ꢀ) were
used. These two cell lines were a kind gift from Dr. Bert Vogelstein
(Johns Hopkins University, Baltimore). HCT116 cells were cultured
in McCoy’s 5A medium supplemented with 10% fetal bovine serum,
penicillin (100 units/mL) and streptomycin (100 lg/mL) at 37 °C in
5.2.2.1. Boc-NH-(CH2)2-1-naphthylamide (7).
White solid
a humid atmosphere of 5% CO2/95% air. For the cell viability assay,
cells were plated at 1 ꢁ 104 into each well of a 96-well microplate
with various concentrations of compounds 1–9. Cell viability was
determined by the WST-1 assay.41
(61.5%), mp 125–130 °C, Rf 0.49 (AcOEt/hexane = 1:1), RT
35.1 min, ESI-MS m/z Calcd [M+H]+ 315.16. Found 315.19. Anal.
Calcd for C18H22N2O3: C, 68.77; H, 7.05; N, 8.91. Found: C, 68.52;
H, 7.24; N, 8.85. 1H NMR (CDCl3) d 7.93 (br s, 1H), 7.86–7.82 (m,
3H), 7.70 (d, J = 8.2 Hz, 1H), 7.51–7.41 (m, 3H), 5.23 (br s, 1H),
3.54 (q, J = 5.8 Hz, 2H), 2.73 (t, J = 5.6 Hz, 2H), 1.44 (s, 9H).
5.5. Colony formation
Reproductive cell death was assayed by measuring colony for-
mation with and without MMS/compound 4. Cells (from 500 up
to 1 ꢁ 104 cells per plate, depending on the dose level) were seeded
per 10 cm dish and treated with vehicle (DMSO), MMS, compound
4, or MMS plus compound 4. Colonies that appeared after 8 days
were fixed with methanol, stained with methylene blue, and
counted. Relative colony formation (%) was expressed as colonies
per treatment level/colonies that appeared in the control.
5.2.2.2. Boc-NH-(CH2)4-1-naphthylamide (8).
White solid
(63.8%), mp 122–127 °C, Rf 0.38 (AcOEt/hexane = 1:1), RT
37.7 min, ESI-MS m/z calcd [M+H]+ 343.19. Found 343.42. Anal.
Calcd for C20H26N2O3: C, 70.15; H, 7.65; N, 8.18. Found: C, 70.07;
H, 7.70; N, 8.19. 1H NMR (CDCl3) d 7.96 (br s, 1H), 7.90–7.84 (m,
3H), 7.68 (d, J = 8.1 Hz, 1H), 7.50–7.42 (m, 3H), 4.70 (br s, 1H),
3.19 (br s-q, J = 5.7 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 1.85–1.77 (m,
2H), 1.62–1.55 (m, 2H), 1.44 (s, 9H).
5.6. Analysis of DNA fragmentation
5.2.2.3. Boc-NH-(CH2)6-1-naphthylamide (9).
White solid
(82.4%), mp 89–94 °C, Rf 0.60 (AcOEt/hexane = 1:1), RT 40.4 min,
Apoptosis was determined using a DNA fragmentation assay.
Total DNA was extracted from the cultured HCT116 p53+/+ and
HCT116 p53ꢀ/ꢀ cells (5 ꢁ 105) treated for 24 h with compound 4
ESI-MS m/z Calcd [M+H]+ 371.23. Found 371.13. Anal. Calcd for
C
22H30N2O3: C, 71.32; H, 8.16; N, 7.56. Found: C, 71.56; H, 8.23;
N, 7.61. 1H NMR (CDCl3) d 7.88–7.84 (m, 3H), 7.80 (br s, 1H),
7.69 (d, J = 8.1 Hz, 1H), 7.50–7.43 (m, 3H), 4.54 (br s, 1H), 3.11
(br s-q, J = 5.4 Hz, 2H), 2.48 (t, J = 7.3 Hz, 2H), 1.80–1.74 (m, 2H),
1.62–1.55 (m, 2H), 1.44 (s, 9H), 1.44–1.35 (m, 6H).
(13.2 lM or 13.7 lM, respectively, based on their LD50 values) fol-
lowing the method of Sambrook et al.42 and 5
lg aliquots were
separated by 1.5% (w/v) agarose gel electrophoresis in 40 mM
Tris-5 mM sodium acetate-1 mM EDTA (pH 7.8) and stained with
ethidium bromide. DNA bands were visualized under ultraviolet
light.
5.3. Pol assays
Mammalian pols with high activities were purified according to
our previous report,36 and standard pol reaction mixtures for pols
5.7. Apoptosis assay using immunofluorescence microscopy
a
and b have been previously described;37,38 those for pol
c
and for
Aliquots of 2.5 ꢁ 104 cells were plated in each well of an 8-well
40
pols d and
respectively; for pols
pol ; and for pols k,
e
are described by Umeda et al.39 and Ogawa et al.
and , the method is the same as for
and TdT, the method is the same as for
chamber slide (Nunc, NY, USA). HCT116 p53+/+ and HCT116 p53ꢀ/ꢀ
g
l
,
i
j
cells were incubated with compound 4 (13.2 lM or 13.7 lM,
a
respectively, based on their LD50 values) for 24 h at 37 °C. TUNEL
is a method for detecting DNA fragmentation by labeling the termi-
nal end of nucleic acids. The percentage of apoptotic cells was
determined using the ApopTag Red In Situ Apoptosis Detection
Kit (CHEMICON, CA, USA). Apoptotic cells were treated with
pol b. For pol reactions, poly(dA)/oligo(dT)18 (A/T, 2/1) and 20-deox-
ythymidine-50-triphosphate (dTTP) were used as the DNA tem-
plate-primer substrate and nucleotide (dNTP, 20-deoxynucleotide-
50-triphosphate) substrate, respectively. For TdT, oligo(dT)18 (30-
OH) and dTTP were used as the DNA primer and nucleotide sub-
strate, respectively.
25 lM etoposide for 5 h at 37 °C. Culture dishes were stained,
and the percentage of apoptotic cells was examined under a fluo-
The novel peptide analogues of somatostatin (i.e., compounds
1–9) were dissolved in dimethyl sulfoxide (DMSO) at various con-
rescence microscope (Olympus IX70; Olympus, Tokyo, Japan).
centrations and sonicated for 30 s. Then 4
l
L aliquots of the solu-
Acknowledgments
tions were mixed with 16 L of each enzyme (final amount 0.05
l
units) in 50 mM Tris–HCl at pH7.5, containing 1 mM dithiothreitol,
50% glycerol (by vol), and 0.1 mM EDTA, and held at 0 °C for
We are grateful for the donation of calf pol
Takemura of Tokyo University of Science (Tokyo, Japan); rat pol
b, human pols d and from Dr. K. Sakaguchi of Tokyo University
of Science (Chiba, Japan); human pol from Dr. M. Suzuki of Na-
goya University School of Medicine (Nagoya, Japan); mouse pol
a from Dr. M.
10 min. These inhibitor-enzyme mixtures (8
16 L of each of the enzyme standard reaction mixtures, and incu-
bated at 37 °C for 60 min. Activity without the inhibitor was con-
lL) were added to
e
l
c
g

I. Kuriyama et al. / Bioorg. Med. Chem. 21 (2013) 403–411
411
18. Miyazaki, A.; Tsuda, Y.; Fukushima, S.; Yokoi, T.; Vantus, T.; Bokonyi, G.; Szabo,
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This study was supported in part by the MEXT (Ministry of Edu-
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Program for the Strategic Research Foundation at Private Universi-
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Young Scientists (B) (No. 23710262) and Scientific Research (C)
(No. 22590111), respectively, from MEXT. Y. M. acknowledges
Grant-in-Aids for Scientific Research (C) (No. 24580205) from
MEXT, Takeda Science Foundation (Japan), and the Nakashima
Foundation (Japan).
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