Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis

Article abstract of DOI:10.1016/j.bmc.2013.01.022

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC₅₀ <5 μM inhibited cellular DNA replication at 10 μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.

Full text of DOI:10.1016/j.bmc.2013.01.022

Bioorganic & Medicinal Chemistry 21 (2013) 1972–1977
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecule inhibitors of PCNA/PIP-box interaction suppress
translesion DNA synthesis
⇑
Marcelo Actis, Akira Inoue, Benjamin Evison, Scott Perry, Chandanamali Punchihewa, Naoaki Fujii
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage
response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be
promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting
with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor
of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created
T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited
biochemical PCNA/PIP-box interaction at an IC50 <5 lM inhibited cellular DNA replication at 10 lM as
measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors
inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that
the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased cH2AX
Received 6 December 2012
Revised 2 January 2013
Accepted 11 January 2013
Available online 22 January 2013
Keywords:
PCNA
Protein–protein interaction inhibitor
DNA damage
Translesion DNA synthesis
Chemosensitization
induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that
inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
not to repair DNA damage but tolerate it for replicating DNA be-
yond the damage. TLS is executed by specialized DNA polymerases
Therapeutics targeting non-oncogenic mediators that ubiqui-
tously support cancers have been successful.1 Proliferating cell nu-
clear antigen (PCNA) is an essential DNA clamp loader that
organizes numerous components of machinery for DNA replica-
tion, DNA damage repair, chromatin maintenance, and cell cycle
(known as TLS Pols) that differ from those of non-TLS replication
such as Pold.6 PCNA is essential for TLS by recruiting Y-family TLS
Pols to the replication fork.7
We anticipated that, if the pharmacologic effects of T2AA are
based on the biochemical PCNA/PIP-box interaction inhibition,
biochemically active T2AA analogues would also inhibit DNA rep-
lication and TLS as long as they penetrate into chromatin. To verify
this, we generated diverse T2AA analogues and determined their
structure–activity relationship (SAR) in the biochemical PCNA
inhibition and cellular activities for inhibiting DNA replication
and TLS.
progression.2 Given its diverse functions, PCNA can be
a
non-oncogenic molecule supporting cancer growth,3 and thus
inhibitors of PCNA could be useful for cancer therapeutics. Most
PCNA interacting components bind to a cavity of PCNA with a short
sequence motif called a PCNA interacting protein (PIP)-box.2 The
PCNA/PIP-box interaction is absolutely essential for diverse PCNA
functions, and thus compounds inhibiting this interaction have
been expected to inhibit PCNA functions.4
2. Materials and methods
We recently found a novel small molecule inhibitor of the
PCNA/PIP-box interaction. This compound, T2AA, inhibits interac-
tion of PCNA with DNA polymerase d (Pold) on chromatin, sup-
presses genomic DNA replication and translesion DNA synthesis
(TLS) in mammalian cells.5 TLS is a mechanism that enables cells
2.1. Reagents and cell cultures
All chemicals were purchased from Sigma–Aldrich (St. Louis,
MO) and used as received. Sources for plasmids are as indicated
in Acknowledgments. U2OS and HeLa cells were obtained from
American Type Culture Collection (Manassas, VA). GM14931 XP-
G fibroblasts were from Coriell Cell Repositories (Camden, NJ). All
cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum at 37 °C in a humidi-
fied, 5% carbon dioxide incubator.
Abbreviations: FL, firefly luciferase; FP, fluorescence polarization; PCNA, prolif-
erating cell nuclear antigen; PIP, PCNA interacting protein; Pold, DNA polymerase d;
RL, Renilla luciferase; SAR, structure–activity relationship; TLS, translesion DNA
synthesis.
⇑
Corresponding author. Tel.: +1 901 595 5854; fax: +1 901 595 5715.
E-mail address: naoaki.fujii@stjude.org (N. Fujii).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.022

M. Actis et al. / Bioorg. Med. Chem. 21 (2013) 1972–1977
1973
2.2. Preparation of new compounds
EDTA and 300 mM NaOH) for 1 h and then subjected to electropho-
resis (30 V) for 30 min at 4 °C. Neutralization buffer (500 mM Tris–
HCl, pH 7.5) was subsequently added and then washed away with
2 ꢀ 10 min PBS (pH 7.4) washes. Samples were subsequently
See Supplementary data for detail. All chemical compounds as-
sayed were prepared as 10 mM DMSO stock solutions.
stained twice with 2.5 lg/mL propidium iodide for 5 min each,
2.3. Fluorescence polarization assay
rinsed with Milli-Q water and visualized using epi-fluorescence
microscopy. DNA damage was visually measured using the scoring
system as described.8 At least 90 cells were scored per slide.
The fluorescence polarization (FP) assay method was reported
previously.5 Briefly, a solution consisting of 100–200 nM recombi-
nant human PCNA protein and 50 nM N-term 5-carboxyfluores-
cein–labeled PL peptide (SAVLQKKITDYFHPKK)4 in FP buffer
(35 mM HEPES, pH 7.4, 10% glycerol, and 0.01% triton X-100) was
2.7. Cell growth assay
U2OS cells (3 ꢀ 103 cells/well) were cultured with a mixture of
incubated with serial dilutions of test compounds (10–0.5
lM final
indicated cisplatin and each compound in indicated concentrations
concentration) in 96-well polypropylene plates for 0.5 h. FP signals
in millipolarization units for the samples were recorded using an
EnVision plate reader (PerkinElmer, Waltham, MA) after they were
transferred in triplicate into a 384-well black opaque plate. Each
dose–response curve was fitted, and IC50 values were determined
using Prism Software (GraphPad, San Diego, CA).
(40
l
L per well in DMEM) for 4 days in a 384-well plate. Alamar-
L per well) was added,
blue reagent (Invitrogen, Carlsbad, CA; 4
l
and signals were read according to the manufacturer’s recommen-
dation. Signals of wells containing no cells and cells with no drugs
(DMSO) served as 0% and 100% normalization, respectively.
3. Results
2.4. BrdU incorporation and cH2AX induction assays
Cells were cultured at 4 ꢀ 105 cells/well in 12-well plates in
1 mL of medium, treated with drugs as indicated for 18 h, tryp-
sin-lifted, and stained for each assay. BrdU incorporation was mea-
sured with an APC BrdU Flow Kit (BD Pharmingen, San Diego, CA)
following the manufacturer’s instructions after cells were pulsed
3.1. Structure–activity relationship in biochemical inhibition of
PCNA/PIP-box interaction
Using fluorescent polarization competition screening against
the interaction of PCNA protein and a PIP-box peptide, we previ-
ously found that 3,5,30-triiodo thyronine (T3, 1) binds to a cavity
of PCNA to which the PIP-box sequence binds.5 We started a SAR
investigation by changing the iodine substitution pattern and ami-
no acid moiety of 1. As expected from the co-crystal structure of
PCNA/1, in which the presence of two iodines at the 3- and 5-posi-
tions is essential for PCNA binding, removing one of the 3- and 5-
iodines (2) or adding 50-iodine (T4, 3) to 1 abolished the activity,
but removing the 30-iodine (T2, 4) did not. When the amino acid
moiety of 1 was simplified as an acetic acid (5) or propionic acid
(6), the activity was decreased but less than 10 times. Similarly,
in the conversion of 1 to 4, removing the 30-iodine from 6 did
not reduce the activity (7).
with 20 lM BrdU for 15 min right before the trypsin-lifting. cH2AX
was measured with FlowCellect H2A.X DNA Damage kit (Millipore,
Billerica, MA; FCCH025142) according to the manufacturer’s rec-
ommendation, in which the cytometry gating was defined by sam-
ples from DMSO treatment and etoposide (100
as negative and positive controls. All samples were filtered through
40 m nylon mesh prior to flow cytometry. The flow data were ac-
lM, 2 h) treatment
l
quired using an LSRII flow cytometer equipped with 405, 488, 561,
and 640 nm lasers (Becton Dickinson, San Jose, CA).
2.5. TLS assay by plasmid reactivation
GM14931 cells were cultured in DMEM (4 ꢀ 105 cells) and
transfected by a mixture of the pGL4.5 (80 ng) or Pt-pGL4.5
(400 ng, Supplementary data for preparation) and pRL-TK (Prome-
ga, 1.8 lg) using Opti-MEM and FuGene6 (Roche Applied Science,
Penzberg, Upper Bavaria, Germany) according to the manufac-
We next investigated SAR of the acid portion of the scaffolds by
diversifying 5–7 as amides. The activity is sharply decreased as the
size and hydrophobicity of the amide substitution are increased
(9–13). In contrast, the activity was maintained when a hydrophilic
group was substituted on the amide (14–21). T2 amino alcohol
(T2AA, 22) was previously designed by changing the carboxylate
of 4 to an alcohol. We investigated the SAR also by modification
of 22. Addition of a small substituent on the amino group of 22
slightly reduced the potency (23–26). In contrast, addition of a sub-
stituent on the alcohol group position of 22 did not affect the po-
turer’s recommendation. After 5 h, the cells were trypsin-lifted
and replated into a 96-well plate (4 ꢀ 103 cells/100
l
L per well),
and dilution of each test compound (50 lL of 30 lM in DMEM:
i.e., final concentration 10 lM) was added in triplicate. After 72 h
of transfection, culture medium was removed, and activities of fire-
fly luciferase (FL) and Renilla luciferase (RL) were measured by DLR
reagent (Promega) according to the manufacturer’s recommenda-
tion. FL/RL values represent replication of the pGL4.5 or the TLS
product produced from the Pt-pGL4.5, thus showing activities of
SV40-mediated replication and TLS in the cells treated by the com-
pounds, which are indicated as 1 for that of cells treated by DMSO.
tency, even when
a relatively large substitution group was
introduced (27–31) (Fig. 1).
3.2. Inhibition of cellular DNA replication
As PCNA/PIP-box interaction is essential for recruitment of DNA
Pold to the replication fork, inhibitors of the interaction should in-
hibit DNA replication. We previously found that 22 dissociated
PCNA/Pold interaction on chromatin and inhibited DNA replica-
tion.5 To determine whether biochemically active T2AA analogues
also inhibit DNA replication, HeLa cells were treated with each
compound and pulsed with BrdU. The incorporated BrdU was
measured by flow cytometry as the average signal intensity per
population percentage of S-phase cells and normalized to that of
DMSO-treated cells to determine the inhibitory effect for the
2.6. Comet assay
U2OS cells were treated as indicated for 22 h. The cells were
harvested by trypsin, mixed with molten low gelling temperature
agarose (Type VII) and subsequently spread and allowed to set
on a glass slide pre-coated with agarose. Gel-embedded cells were
lysed in ice-cold lysis buffer (100 mM EDTA, 2.5 M NaCl, 10 mM
Tris–HCl, pH 10.5, and 1% Triton X-100) for 1 h and then subjected
to 4 ꢀ 15 min washes with ice-cold Milli-Q water. Samples were
then submerged in chilled alkali electrophoresis buffer (1 mM
DNA replication. In this experimental condition, 10
lM compounds
with the potency of the biochemical PCNA/PIP-box interaction

1974
M. Actis et al. / Bioorg. Med. Chem. 21 (2013) 1972–1977
replication. Therefore, this assay can determine the activity of com-
pounds in inhibiting damage tolerance by TLS and replication of
the plasmid produced after TLS. To determine the activity of com-
pounds in inhibiting plasmid replication and reporter transcription
(but not TLS), we performed the experiments side-by-side using
corresponding intact pGL4. The XP-G fibroblasts were transfected
with either Pt-pGL4 or intact pGL4 and treated with 10
pounds that were active in BrdU incorporation inhibition at
10 M. The net TLS inhibition activity was defined by normalizing
lM com-
l
the reporter signal from Pt-pGL4 to that from intact pGL4. Most of
the compounds tested reduced the normalized signal compared
with that of cells treated by DMSO, suggesting that those com-
pounds inhibit TLS (Fig. 3A).
To ensure that the effect was due to TLS inhibition, we also per-
formed this assay in HeLa cells, which are NER-intact but not SV40-
transformed, thus allowing NER but not replication after TLS. The
compounds showed little or no inhibition of the normalized signal
(Fig. 3B). We also performed the assay in the XP-G fibroblasts using
pGL4 plasmids that had been treated by aqueous acid instead of Pt-
pGL4. Acid hydrolysis induces apurinic/apyrimidinic sites on the
DNA duplex that are repaired as intermediates of base-excision re-
pair (BER).13 Compounds that were active in the TLS inhibition as-
say showed no effect in this assay (Fig. S3). Taken together, these
results suggest that PCNA/PIP-box interaction inhibitors do not af-
fect NER and BER.
3.4. Increasing cisplatin-mediated DNA-damage response
TLS is a DNA damage tolerance process that releases cells from
replication stress by the damage. When a replication fork is stalled
at a DNA lesion, it triggers activation of the ATR pathway, which
causes histone H2AX phosphorylation (cH2AX). This event is very
Figure 1. Structure–activity relationship of the PCNA/PIP-box interaction inhibi-
tors. Chemical structures and IC50 values of the fluorescence polarization (FP)
biochemical competition assay for PCNA protein/FITC-labeled PL peptide interac-
tion are shown for indicated numbered compounds. Preparation and characteriza-
tion data of each compound are described in Supplementary data.
sensitive and responsive to virtually any type of DNA damage,
including not only double-strand breaks but also alkylation and
cross-links by cisplatin. When TLS is inhibited, the damage should
be recognized and
of PCNA/PIP-box interaction inhibitors, HeLa cells were treated
with a combination of cisplatin (1 M) and a PCNA/PIP-box
inhibitor (10 M). The population of H2AX-positive cells was
cH2AX should be induced. To validate this effect
l
inhibition IC50 <5 lM inhibited the BrdU incorporation (Fig. 2),
l
c
suggesting that the DNA replication inhibition is based on the
PCNA/PIP-box interaction inhibition on the chromatin. Some com-
pounds that are active in the biochemical inhibition but not in the
cellular DNA replication (19, 25) most likely failed to penetrate
chromatin and thus were not further evaluated.
measured by flow cytometry to determine the effect of the
PCNA/PIP-box inhibitors on cisplatin-mediated DNA damage
response. Compounds that inhibited TLS increased the
positive population (Fig. 4A). Except few, these compounds did
not significantly increase the H2AX in the absence of cisplatin
cH2AX-
c
(Fig. S4). These results suggest that the PCNA/PIP-box inhibitor
increases the DNA damage response of cells. This effect is similar
to that observed for aphidicolin,14 which induces DNA replication
stress that increases exposure of single-stranded DNA15 vulnerable
to damage induction by cisplatin.
TLS is a terminological definition as a replication process be-
yond a DNA lesion without repairing the damage, therefore inhib-
iting TLS does not affect the amount of DNA damage. To verify this,
cells were charged by cisplatin with or without the PCNA/PIP-box
inhibitor 22. DNA damage left in the genomic DNA after the treat-
ment was directly measured by a comet assay. The level of comet
tail induced by the cisplatin was not significantly affected by 22
3.3. Inhibition of replication-dependent translesion DNA
synthesis
TLS enables cells to bypass a damaged DNA lesion without
repairing it. TLS is executed by polymerases that are specialized
for the TLS process. As replication DNA polymerases such as Pold
do, Y-family TLS Pols interact with PCNA by their PIP-box,9 which
is essential for TLS activation. Therefore, one possible pharmaco-
logic effect of a PCNA/PIP-box interaction inhibitor is TLS inhibi-
tion. To verify this, we assayed compounds for inhibition of TLS
by reactivation of damaged plasmids.10 The pGL4 firefly lucifer-
ase-reporter plasmid contains an SV40 replication origin se-
quence11 in its SV40 early enhancer/promoter region (Fig. S2)
and thus can be replicated in SV40-transformed cells. The pGL4
was treated with activated cisplatin to globally induce damages
of an amount that can be reactivated in cells. The damaged plasmid
(Pt-pGL4) was transfected into SV40-transformed GM14931 XP-G
human fibroblasts. The GM14931 cells has a severe truncation of
the XPG protein, which is essential for nucleotide excision repair
(NER);12 thus, the damage to the plasmid in this cell line is
not NER-repaired but TLS-tolerated to allow SV40-dependent
(Fig. 4B); however, cH2AX was induced (Fig. 4A). Taken together,
these results suggest that a PCNA/PIP-box inhibitor does inhibit
TLS more significantly than DNA repair processes (Fig. 3B).
3.5. Increasing suppression of cell proliferation by cisplatin
As we observed the effect of PCNA/PIP-box interaction inhibi-
tors on cisplatin-induced DNA damage response, we assayed the
growth suppression effect of co-treatment of cells by cisplatin

M. Actis et al. / Bioorg. Med. Chem. 21 (2013) 1972–1977
1975
Figure 2. The PCNA/PIP-box interaction inhibitors block DNA replication. Relationship of IC50 potency in the FP assay (white bars and right y-axis) and activity of BrdU
incorporation inhibition assay for 10
potency.
lM treatment of each indicated compound in HeLa cells (gray bars and left y-axis) are shown. The data are sorted by the order of the FP
Figure 3. The PCNA inhibitors block reactivation of a cisplatin-damaged plasmid in GM14931 but not in HeLa cells. GM14931 or HeLa cells were transfected by cisplatin-
damaged Pt-pGL4 or intact pGL4 and pRK-TK plasmids and treated with 10 M indicated compound in triplicate. Activity of the firefly luciferase was normalized to that of
l
Renilla luciferase, and the normalized values were further normalized by the average of that of DMSO-treated wells as 1. The effect in the TLS (GM14931, A) or NER (HeLa, B)
was determined by dividing the DMSO-normalized values for Pt-pGL4 by that for intact pGL4 in each sample, which is shown in the figures. Compound 21, a weak inhibitor
for the PCNA/PIP-box interaction and the DNA replication (Fig. 2) serves as a negative control.
and the inhibitors. U2OS cells are relatively resistant to cisplatin
but have wild-type p53 and thus can respond to DNA damage by
activating the ATM-p53 pathway.16 Proliferation of U2OS cells
was little affected by single treatment with a PCNA inhibitor
(Fig. 5A) or cisplatin (Fig. 5B) up to 15 lM. In contrast, the reduc-
tion of proliferation by cisplatin titration was significant in the
presence of the inhibitors, showing the sensitization effect of the
inhibitor in these cells.

1976
M. Actis et al. / Bioorg. Med. Chem. 21 (2013) 1972–1977
Figure 4. The PCNA inhibitors enhance DNA damage response without increasing DNA damage by cisplatin. (A) The PCNA inhibitors enhance cisplatin-induced histone H2AX
phosphorylation. HeLa cells were treated with cisplatin (1 M) and the indicated compound (10 M) for 18 h. The percentage of H2AX-positive population was quantified by
flow cytometry. Change of H2AX level for each sample is indicated relative to the H2AX-positive population of the cisplatin/DMSO-treated cells. (B) The PCNA inhibitor
does not further increase cisplatin-induced genomic DNA damage. U2OS cells were treated with cisplatin (25 M), 22 (25 M), or combination of cisplatin (25 M) and 22
(25 M), for 22 h. Cell electrophoresis and comet tail scoring were done as described in Section 2. The comet tail score for each treatment is shown. NT: no treatment.
l
l
c
c
c
l
l
l
l
4. Discussion
In our previous study, we observed that the two iodines at the
3- and 5-positions, but not the amino acid moiety, of 1 contact
the PCNA cavity that adopts a PIP-box sequence.5 The SAR observa-
tion in this study is well matched to this previous structural obser-
vation, especially for changing the substitution pattern of the
iodines. Modification of the acid moiety of 5 and 7 or alcohol moi-
ety of 22 yielded a series of active compounds, which is consistent
with the concept that the corresponding region of 1 does not di-
rectly contact PCNA. However, the observation that a hydrophobic
group (10–13) is not tolerated for the acid substitution is puzzling,
given that structurally similar hydrophilic variants (14, 16, 18, 21)
are active. This suggests that the substitution at this region could
induce a steric conflict with the PCNA protein surface, but a polar
group in the substituent affords an unidentified additional interac-
tion site that compensates for the affinity loss by the conflict. If this
is true, more potent new inhibitors could be further generated by
optimizing the hydrophilic substituent. However, as high hydro-
philicity of a compound decreases its cell membrane permeability,
optimization by this strategy should be undertaken only after care-
fully considering the trade-off between biochemical potency (FP
assay) and cellular activity (BrdU and TLS assays).
Figure 5. The PCNA inhibitors enhance cisplatin-induced growth suppression.
U2OS cells were cultured with (A) the indicated concentrations of each compound,
or (B) cisplatin and the indicated compound (15 lM) for 4 days. Viability of the cells
was measured by Alamarblue reagent, and signals were normalized to those of no
cell well (0%) and cells with no drugs (100%). Error bars represent SD.
PIP-box binding cavity, our observation supports the idea that tar-
geting the PIP-box site is sufficient for TLS inhibition.
TLS is a two-step process in which insertion of a nucleotide be-
yond a lesion and extension from the lesion are executed by differ-
ent sets of TLS Pols.27,28 Which step was inhibited by the PCNA
inhibitors? In the two-step model, TLS Pols for the insertion step
The PIP-box interaction is essential for numerous PCNA func-
tions.2 In addition, recent studies have shown that monoubiquiti-
nation of PCNA is also important for some PCNA functions,
specifically for activation of TLS by polymerase switching.17–19
When cells receive DNA damage such as cisplatin treatment, PCNA
are Polg, Poli, and Polj, all of which are Y-family Pols containing
a PIP-box. The TLS Pol for the extension step is regarded as almost
always Polf, which is switched from the insertion TLS Pol on PCNA
by being bridged with REV1. According to this model, only the
insertion step could be suppressed by inhibiting the PCNA/PIP-
box interaction because neither Polf nor REV1 has a PIP-box. How-
ever, REV1 binds to PCNA in a region different from the PIP-box
adopting site29,30 and recruits the REV3L subunit of Polf by direct
binding;31,32 thus, the extension step could be indirectly affected
by PCNA inhibitors because Polf must be successively switched
back to Pold on PCNA to resume regular DNA replication.33 Rescue
experiments could determine which TLS Pols are functionally
inhibited by the inhibitors in cells.
In the NER process, XPG protein executes dual-incision to re-
move the lesion. XPG has a PIP-box; thus, PCNA has been consid-
ered to support recruitment of XPG to the lesion.34 However, we
did not observe a significant effect of our PCNA inhibitors in the
NER of a cisplatin-damaged plasmid DNA in HeLa cells. A study
is monoubiquitinated.20 It is suggested that a TLS Pol such as Pol
g
binds to non-ubiquitinated PCNA via PIP-box interaction with
modest affinity, but when PCNA is monoubiquitinated, the ubiqui-
tin moiety becomes an additional binding site to the TLS Pol via its
UBZ domain, increasing the affinity as a whole and promoting TLS.
However, the relative importance of the PIP-box interaction and
UBZ domain interaction is unclear. It has been somewhat contro-
versial whether monoubiquitination is absolutely essential for
TLS.21–25 Actually, the biochemical affinity of the UBZ domain of
Pol
PCNA/Pol
box interaction inhibitors can suppress TLS and abolish Pol
g
to a ubiquitin is reportedly much weaker26 than that of
PIP-box interaction.9 Our observations that PCNA/PIP-
g
g
recruitment to chromatin PCNA foci suggest that a PIP-box is abso-
lutely essential for TLS and targeting it is sufficient to inhibit TLS.
As our compounds very unlikely interfere with the monoubiquiti-
nated PCNA site (Lys164) that is located 30–35 Å away from the

M. Actis et al. / Bioorg. Med. Chem. 21 (2013) 1972–1977
1977
that investigated NER mechanism in the incision step showed that
References and notes
XPG protein is recruited to the local UV-damaged lesion before
PCNA is recruited there to form a complex.35 Therefore, XPG may
not need PCNA for being loaded to the lesion, and thus PCNA inhib-
itors may have not inhibited NER.
1. Luo, J.; Solimini, N. L.; Elledge, S. J. Cell 2009, 136, 823.
2. Moldovan, G. L.; Pfander, B.; Jentsch, S. Cell 2007, 129, 665.
3. Stoimenov, I.; Helleday, T. Biochem. Soc. Trans. 2009, 37, 605.
4. Kontopidis, G.; Wu, S. Y.; Zheleva, D. I.; Taylor, P.; McInnes, C.; Lane, D. P.;
Fischer, P. M.; Walkinshaw, M. D. Proc. Natl. Acad. Sci. U.S.A. 1871, 2005, 102.
5. Punchihewa, C.; Inoue, A.; Hishiki, A.; Fujikawa, Y.; Connelly, M.; Evison, B.;
Shao, Y.; Heath, R.; Kuraoka, I.; Rodrigues, P.; Hashimoto, H.; Kawanishi, M.;
Sato, M.; Yagi, T.; Fujii, N. J. Biol. Chem. 2012, 287, 14289.
6. Waters, L. S.; Minesinger, B. K.; Wiltrout, M. E.; D’Souza, S.; Woodruff, R. V.;
Walker, G. C. Microbiol. Mol. Biol. Rev. 2009, 73, 134.
7. Sale, J. E.; Lehmann, A. R.; Woodgate, R. Nat. Rev. Mol. Cell Biol. 2012, 13, 141.
8. Collins, A. R. Mol. Biotechnol. 2004, 26, 249.
9. Hishiki, A.; Hashimoto, H.; Hanafusa, T.; Kamei, K.; Ohashi, E.; Shimizu, T.;
Ohmori, H.; Sato, M. J. Biol. Chem. 2009, 284, 10552.
10. Cenni, B.; Kim, H. K.; Bubley, G. J.; Aebi, S.; Fink, D.; Teicher, B. A.; Howell, S. B.;
Christen, R. D. Br. J. Cancer 1999, 80, 699.
11. Tseng, B. Y.; Ahlem, C. N. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2342.
12. Vermeulen, W.; Jaeken, J.; Jaspers, N. G.; Bootsma, D.; Hoeijmakers, J. H. Am. J.
Hum. Genet. 1993, 53, 185.
5. Conclusion
Non-oncogene addiction of cancers justifies targeting ubiquitous
mediators for cancer therapeutics.1 One common concern regarding
targeting non-oncogenic molecules is nonspecific toxicity to normal
tissues, as seen in traditional chemotherapies. It is possible to selec-
tively eliminate cancers by targeting non-oncogenic molecules if
they are more active in cancers or have a different mode of action.
PCNA is a multifunctional molecule that is included in virtually
any DNA replication and repair processes;2 thus, inhibiting PCNA
has been expected to be highly toxic. However, we previously found
that T2AA 22 is not promiscuously cytotoxic but just cytostatic,5
justifying PCNA inhibition as a strategy to target cancers36 as an
adjuvant therapeutic with DNA damaging agents.
13. McCullough, A. K.; Dodson, M. L.; Lloyd, R. S. Annu. Rev. Biochem. 1999, 68, 255.
14. Sargent, J. M.; Elgie, A. W.; Williamson, C. J.; Taylor, C. G. Br. J. Cancer 1996, 74,
1730.
15. Vassin, V. M.; Anantha, R. W.; Sokolova, E.; Kanner, S.; Borowiec, J. A. J. Cell Sci.
2009, 122, 4070.
16. Canman, C. E.; Chen, C. Y.; Lee, M. H.; Kastan, M. B. Cold Spring Harb. Symp.
Quant. Biol. 1994, 59, 277.
Mechanistically, PCNA inhibitors cannot selectivity target TLS
without affecting regular replication, as we saw that they are
inhibitory in both BrdU and TLS assays. This is one limitation of
PCNA inhibitors. Cancer selectivity of traditional chemotherapy is
based on the difference of cell growth properties between cancer
and normal cells, in which the majority of normal cells are cell-cycle
arrested. This principle could also be applied for PCNA inhibitors
because PCNA supports cell cycle progression by supporting DNA
replication. In addition, PCNA inhibitors could selectively target
replication-dependent TLS by the polymerase-switching process.
TLS is important for maintaining the genomic stability of normal
tissues.37,38 This TLS has to be executed in non-replicative cells
outside the context of the replication fork, thus being regarded as
a gap-filling mechanism39 that is replication-independent and thus
less PCNA-dependent. If a PCNA inhibitor can selectively inhibit
replication-dependent TLS over replication-independent TLS, it
could be validated as a novel chemotherapeutic adjuvant. We
assayed our PCNA inhibitors in SV40-dependent replication cou-
pled by TLS, which indicates inhibition in replication-dependent
TLS, but not in NER, whose mechanism is gap-filling that is remi-
niscent of replication-independent TLS. To further evaluate this
mechanistic TLS selectivity, more TLS assays need be performed
next for replication-independent TLS using a gapped plasmid in
which the gap contains a lesion.28,40
17. Masuda, Y.; Piao, J.; Kamiya, K. J. Mol. Biol. 2010, 396, 487.
18. Bienko, M.; Green, C. M.; Sabbioneda, S.; Crosetto, N.; Matic, I.; Hibbert, R. G.;
Begovic, T.; Niimi, A.; Mann, M.; Lehmann, A. R.; Dikic, I. Mol. Cell 2010, 37, 396.
19. Freudenthal, B. D.; Gakhar, L.; Ramaswamy, S.; Washington, M. T. Nat. Struct.
Mol. Biol. 2010, 17, 479.
20. Hoege, C.; Pfander, B.; Moldovan, G. L.; Pyrowolakis, G.; Jentsch, S. Nature 2002,
419, 135.
21. Hendel, A.; Krijger, P. H.; Diamant, N.; Goren, Z.; Langerak, P.; Kim, J.; Reissner,
T.; Lee, K. Y.; Geacintov, N. E.; Carell, T.; Myung, K.; Tateishi, S.; D’Andrea, A.;
Jacobs, H.; Livneh, Z. PLoS Genet. 2011, 7, e1002262.
22. Krijger, P. H.; van den Berk, P. C.; Wit, N.; Langerak, P.; Jansen, J. G.; Reynaud, C.
A.; de Wind, N.; Jacobs, H. DNA Repair (Amst.) 2011, 10, 1051.
23. Acharya, N.; Brahma, A.; Haracska, L.; Prakash, L.; Prakash, S. Mol. Cell. Biol.
2007, 27, 7266.
24. Acharya, N.; Yoon, J. H.; Gali, H.; Unk, I.; Haracska, L.; Johnson, R. E.; Hurwitz, J.;
Prakash, L.; Prakash, S. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17724.
25. Acharya, N.; Yoon, J. H.; Hurwitz, J.; Prakash, L.; Prakash, S. Proc. Natl. Acad. Sci.
U.S.A. 2010, 107, 10401.
26. Bomar, M. G.; Pai, M. T.; Tzeng, S. R.; Li, S. S.; Zhou, P. EMBO Rep. 2007, 8, 247.
27. Johnson, R. E.; Washington, M. T.; Haracska, L.; Prakash, S.; Prakash, L. Nature
2000, 406, 1015.
28. Shachar, S.; Ziv, O.; Avkin, S.; Adar, S.; Wittschieben, J.; Reissner, T.; Chaney, S.;
Friedberg, E. C.; Wang, Z.; Carell, T.; Geacintov, N.; Livneh, Z. EMBO J. 2009, 28,
383.
29. Guo, C.; Sonoda, E.; Tang, T. S.; Parker, J. L.; Bielen, A. B.; Takeda, S.; Ulrich, H.
D.; Friedberg, E. C. Mol. Cell 2006, 23, 265.
30. Sharma, N. M.; Kochenova, O. V.; Shcherbakova, P. V. J. Biol. Chem. 2011, 286,
33557.
31. Kikuchi, S.; Hara, K.; Shimizu, T.; Sato, M.; Hashimoto, H. J. Biol. Chem. 2012,
287, 33847.
32. Wojtaszek, J.; Lee, C. J.; D’Souza, S.; Minesinger, B.; Kim, H.; D’Andrea, A. D.;
Walker, G. C.; Zhou, P. J. Biol. Chem. 2012, 287, 33836.
33. Baranovskiy, A. G.; Lada, A. G.; Siebler, H. M.; Zhang, Y.; Pavlov, Y. I.; Tahirov, T.
H. J. Biol. Chem. 2012, 287, 17281.
Acknowledgments
We thank Ms. Michele Connelly for laboratory support; Dr. Ann
Marie Hamilton-Easton for flow cytometry support; Mr. Brett
Waddell for attempting SPR measurement; Dr. Alex Sparreboom
and Ms. Alice Gibson for attempting quantification of cisplatin
accumulation in genomic DNA; and Mr. David Galloway for gram-
matical advice and preparation of this manuscript. This study was
financially supported by the American Lebanese Syrian Associated
Charities (ALSAC) and American Cancer Society Research Scholar
Grant #RSG CDD-120969 (N.F.).
34. Kannouche, P.; Broughton, B. C.; Volker, M.; Hanaoka, F.; Mullenders, L. H.;
Lehmann, A. R. Genes Dev. 2001, 15, 158.
35. Mocquet, V.; Laine, J. P.; Riedl, T.; Yajin, Z.; Lee, M. Y.; Egly, J. M. EMBO J. 2008,
27, 155.
36. Stoimenov, I.; Helleday, T. Biochem. Soc. Trans. 2009, 37, 605.
37. Lange, S. S.; Wittschieben, J. P.; Wood, R. D. Nucleic Acids Res. 2012, 40, 4473.
38. Wittschieben, J. P.; Reshmi, S. C.; Gollin, S. M.; Wood, R. D. Cancer Res. 2006, 66,
134.
39. Lopes, M.; Foiani, M.; Sogo, J. M. Mol. Cell 2006, 21, 15.
40. Hashimoto, K.; Cho, Y.; Yang, I. Y.; Akagi, J.; Ohashi, E.; Tateishi, S.; de Wind, N.;
Hanaoka, F.; Ohmori, H.; Moriya, M. J. Biol. Chem. 2012, 287, 9613.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.022.