Antiproliferative and apoptotic activities of sequence-specific histone acetyltransferase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.06.037

In parallel to monomeric epigenetic regulators, sequence-specific epigenetic regulators represent versatile synthetic dual-target ligands that achieve regulatory control over multi-gene networks. Development of DNA-binding domain (DBD)-HDAC inhibitors and DBD-HAT activators, which result in increased histone acetylation, has become one promising research field. However, there is no report regarding the gene regulatory pattern by sequence-specific epigenetic repressor. We report here for the first time, the synthesis of DBD-HAT inhibitors and demonstrate that these conjugates could retain their dual-target activity using predicted working model of thermal stability assay and in vitro HAT activity assay. Evaluation of antiproliferative activity in cancer cells showed that 2 (with a medium linker length of 13-atom) exhibited the highest antiproliferative activity in p53 wild-type cancer cell lines (IC₅₀ of 1.8–2.6 μM in A549 and MV4-11 cells) and not in p53 mutant cancer cell lines. A mechanistic investigation using microarray analysis and an apoptotic assay showed that the antiproliferative effect of 2 occurred via the up-regulation of p53 target genes, and the subsequent initiation of p53-dependent apoptosis. Our research on sequence-specific dual-target epigenetic repressor offers us an alternative way to modulate HAT-governed therapeutically important genes and contributes to offer a fresh insight into antitumor therapeutics.

Full text of DOI:10.1016/j.ejmech.2017.06.037

Accepted Manuscript
Antiproliferative and apoptotic activities of sequence-specific histone
acetyltransferase inhibitors
Zutao Yu, Junichi Taniguchi, Yulei Wei, Ganesh N. Pandian, Kaori Hashiya,
Toshikazu Bando, Hiroshi Sugiyama
PII:
S0223-5234(17)30483-X
10.1016/j.ejmech.2017.06.037
EJMECH 9532
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2017
Revised Date: 30 May 2017
Accepted Date: 22 June 2017
Please cite this article as: Z. Yu, J. Taniguchi, Y. Wei, G.N. Pandian, K. Hashiya, T. Bando, H.
Sugiyama, Antiproliferative and apoptotic activities of sequence-specific histone acetyltransferase
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.037.
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ACCEPTED MANUSCRIPT
Antiproliferative and Apoptotic Activities of Sequence-Specific
Histone Acetyltransferase Inhibitors
Zutao Yua, Junichi Taniguchia, Yulei Weia, Ganesh N. Pandianb, Kaori Hashiyaa, Toshikazu
Bandoa*, Hiroshi Sugiyamaa,b*
aDepartment of Chemistry,
Graduate
School
of
Science,
Kyoto
University
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan)
bInstitute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University
Yoshida-Ushinomiyacho, Sakyo-ku, Kyoto 606-8501 (Japan).
ABSTRACT
In parallel to monomeric epigenetic regulators, sequence-specific epigenetic regulators represent
versatile synthetic dual-target ligands that achieve regulatory control over multi-gene networks.
Development of DNA-binding domain (DBD)-HDAC inhibitors and DBD-HAT activators, which
result in increased histone acetylation, has become one promising research field. However, there is
no report regarding the gene regulatory pattern by sequence-specific epigenetic repressor. We
report here for the first time, the synthesis of DBD-HAT inhibitors and demonstrate that these
conjugates could retain their dual-target activity using predicted working model of thermal
stability assay and in vitro HAT activity assay. Evaluation of antiproliferative activity in cancer
cells showed that 2 (with a medium linker length of 13-atom) exhibited the highest
antiproliferative activity in p53 wild-type cancer cell lines (IC50 of 1.8-2.6 µM in A549 and
MV4-11 cells) and not in p53 mutant cancer cell lines. A mechanistic investigation using
microarray analysis and an apoptotic assay showed that the antiproliferative effect of 2 occurred
via the up-regulation of p53 target genes, and the subsequent initiation of p53-dependent apoptosis.
Our research on sequence-specific dual-target epigenetic repressor offers us an alternative way to
modulate HAT-governed therapeutically important genes and contributes to offer a fresh insight
into antitumor therapeutics.
Keywords:
Epigenetics; Sequence specificity; Polyamide; HAT inhibitor; Antiproliferation; Apoptosis
Abbreviations: HAT, histone acetyltransferase; DBD, DNA-binding domain; HDAC, histone
deacetylase; PIP, pyrrole-imidazole polyamides; DMSO, dried dimethylsulfoxide; IPA, ingenuity
pathway analysis; GSEA, gene set enrichment analysis; KEGG, Kyoto encyclopedia of genes and
genomes; ESI-TOF MS, electrospray ionization time-of-flight mass spectrometry, DMF,
dimethylformamide; HPLC, high performance liquid chromatography; FBS, fetal bovine
serum;
*Corresponding author. Department of Chemistry, Graduate School of Science, Kyoto University
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan)
E-mail addresses: bando@kuchem.kyoto-u.ac.jp (T. Bando), hs@kuchem.kyoto-u.ac.jp (H.
Sugiyama).

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1. Introduction
Among epigenetic families, histone acetylation has been identified as a key histone marker that
switches on gene expression, ant its dysregulation are associated with substantial diseases [1].
Several epigenetic agents, such as histone deacetylase (HDAC) inhibitors (SAHA and TSA),
histone acetyltransferase (HAT) activators (CTB and CTPB), and HAT inhibitors (C646 and
anacardic acid), have been approved or are in preclinical development as they showed promising
therapeutic efficacy [2-5]. Along with the extensively studied monomeric epigenetic regulators,
sequence-specific epigenetic activators have recently gained prominence as an emerging class of
versatile synthetic dual-target ligands capable of having regulatory control over multi-gene
networks (Figure 1) [6, 7].
Figure 1. Chemical structures of the HDAC inhibitors SAHA and SAHA-I, HAT activators CTB and CTB-I, and
HAT inhibitor C646.
Our lab has focused on sequence-specific epigenetic activators that involve conjugation of
epigenetic modulators with DNA-binding domains (DBDs), i.e., pyrrole-imidazole polyamides
(PIPs) [8]. PIPs are programmable DNA minor groove binders with the ability to recognize and
bind specific DNA sequences strongly (with the Py/Py pair recognizing A/T or T/A, Py/Im
recognizing C/G, and Im/Py recognizing G/C) [9]. By incorporating a variable 8-ring PIP moiety,
compound library of both HDAC inhibitor conjugates (SAHA-PIP) [10], we showed gene-specific
histone acetylation and downstream gene activation in living cells. Promisingly, after conjugation
with SAHA, the PIP-I sequence (DNA-binding sequence, 5′–WWCCWW–3′, W = A/T) exhibited
the highest potential to upregulate SOX2, OCT4 and NANOG, which are responsible for iPS cell
formation and maintenance (Figure 1) [11]. More interestingly, when SAHA was replaced with
CTB, CTB-I showed a gene activation pattern that was similar to that of SAHA-I, although it
targeted a different epigenetic regulator [12].
In contrast with sequence-specific epigenetic activators, such as SAHA-PIP and CTB-PIP which
functionally resulted in gene activation, no sequence-specific epigenetic repressor has been reported.
In this work, we constructed dual-function and sequence-specific HAT inhibitor via C646
conjugation with PIP-I, for the development of novel cancer therapies [5]. Through independent
lines of evidence, we characterize the activity mode of sequence-specific HAT inhibitor conjugates
and demonstrate the anti-proliferative activities in cancer cell lines including A549 and MV4-11
cells. Microarray analysis and an apoptotic assay revealed the initiation of apoptosis caused by the
upregulation of p53 target genes as the mechanism behind the antiproliferative effect of in cancer

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cells. Because HAT enzymes are known to regulate the genes associated with tumorigenesis, our
study suggests the novel chemical approach to modulate the gene-regulatory pathway in a
sequence-specific manner.
2. Results and discussion
2.1 Design and synthesis
C646, which was reported by Philip A. Cole through silica screening, is the most potent HAT
inhibitor available currently. C646 inhibits p300 with a Ki value of 400 nM in vitro and significantly
downregulates histone acetylation in human lung cancer cells and melanoma cells [5]. C646
contains a carboxyl group, and its amide analogs or propargylamine conjugates exhibit the same
level of HAT inhibitory activity, which implies that PIP conjugation at the carboxyl site is a good
choice, while maintaining HAT inhibitory activity [13].
Scheme 1. Structural design of PIP conjugates of the HAT inhibitor C646 (C646-PIP).
Inspired by the 8-ring PIP-I, we performed PIP-I conjugation with the HAT inhibitor C646
(C646-I) [12]. The linker region between the functional group and PIP might affect its binding
affinity and functional effect. Our group reported six kinds of linkers of SAHA-PIP, and revealed
that a linker length of 12-atom yielded maximum expression of OCT3/4 [14]. In this work, after
taking linker length and chain flexibility into consideration, we designed C646-I conjugates with
three types of linkers, i.e., a shorter linker (8-atom, 1), a moderate linker (13-atom, 2), and a longer
linker (18-atom, 3) (Scheme 1). Concomitantly, an alternative PIP sequence ligated with C646 (4)
was also designed, to investigate the PIP sequence/activity relationship.
C646-PIP synthesis started from pyrrole-oxime resin [15]. Using an 11-step Fmoc-protected
solid-phase synthesis, four PIPs were synthesized and cleaved by N,N-dimethylamine. After
purification using a reverse-phase flash column, our target compounds were synthesized by
combining the terminal amino group of the PIP chain with commercially available C646, which
contains a carboxyl group, with the help of the coupling agent PyBOP (Supplementary Data S2).
These conjugates were then purified in high yield.
2.2 Determination of the DNA-binding affinity
A predictive working system in molecular level to evaluate dual-target conjugates would
accelerate the research progress and obviate unintended bioactivity in the early research phase.

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Since C646-PIP conjugates contain DNA binding moiety and HAT inhibitor, we presume that
thermal stability assay and HAT activity assay might be suitable for preliminary evaluation. We
first performed Tm assay to test thermal stabilization of dual-function conjugates-DNA complexes
[16].
Table 1. Results of thermal stability assay
Match sequence
Mismatch sequence
ODN3: 5'-ACTTATCCACTATAGA-3'
ODN4: 3'-TGAATAGGTGATATCT-5'
Tm = 34.3 ℃ (± 0.1)
ODN1: 5'-ACTTATTCCATATAGA-3'
ODN2: 3'-TGAATAAGGTATATCT-5'
Tm = 32.5 ℃ (± 0.1)
Conjugates
Tm/℃
∆Tm/℃a
∆∆Tm/℃
Tm/℃
∆Tm/℃
11.0
∆∆Tm/℃b
—
—
—
—
49.4 (± 0.1)
50.6 (± 0.1)
50.9 (± 0.1)
46.1 (± 0.1)
16.9
45.3 (± 0.1)
45.8 (± 0.1)
46.5 (± 0.1)
55.2 (± 0.1)
5.9
1
2
3
4
18.1
11.5
6.6
18.4
12.2
6.2
13.6
20.9
-7.3
a∆Tm = ∆Tm(dsDNA + PIP) - ∆Tm(dsDNA); b∆∆Tm = ∆Tm(match) - ∆Tm(mismatch)
As shown in Table 1, conjugates 1-3 stabilized match ODN1/2 with ∆Tm of 16.9, 18.1, and 18.4 ℃
respectively. Upon the incubation with mismatch ODN3/4, conjugates 1-3 showed a far weaker
binding affinity (∆Tm of 11.0-12.2 ℃ ), which demonstrated that conjugates 1-3 could
discriminate between match and mismatch sequence with binding sequence specificity (∆∆Tm)
ranged from 5.9- 6.6 ℃. Regarding conjugate 4, it also showed notable binding affinity to its
match ODN3/4 with ∆Tm of 20.9 ℃and sequence specificity of ∆Tm of 7.3 ℃. These results
confirmed that upon the conjugation with C646, PIP retained its high binding affinity and
sequence specificity to targeted DNA sequences.
2.3 In vitro HAT activity assay
After conjugation with PIP, functional moieties, such as SAHA, CTB, and alkylating agents, still
harbored their functional activity toward the primary targets (HDAC inhibition, HAT activation
and DNA alkylation), with only a slight loss of activity [17]. Therefore, it is mandatory that the
conjugation of PIP-I with C646 does not diminish the histone acetylation inhibitory activity. We
then evaluated the HAT activity of all synthesized C646-PIP conjugates using a commercially
available HAT activity kit. We used p300 as the HAT enzyme, as C646 showed high selectivity to
p300 compared with a panel of similar functional enzymes, such as PCAF, GCN5, AANAT, Sas,
Moz, and Rtt109 [5]. The HAT inhibitory activity was assessed using fluorescence absorption on a
microreader using the H3 peptide and Ac-CoA as substrates. Besides, we also designed and
evaluated anacardic acid, a widely used HAT inhibitor, and its conjugates with PIP in a predictive
working system [18]. However, after PIP conjugation, they did not show any HAT inhibitory
activity (Supplementary S3 and S4). The conjugation of PIP blocked the binding of anacardic acid
to the active pocket of p300, which might be responsible for the undesired outcome.

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Figure 2. HAT inhibitory activity assay. The enzymatic activity of HAT was tested in the presence of C646, 1, 2, 3,
4, and PIP-I. Each effector was individually administered at two concentrations (1 and 5 µM) to the p300 enzyme
using a HeLa cell nuclear extract as a positive control and the 0.5% DMSO group as a control group. P<0.05.
Our results showed an IC50 of 2.4 µM for C646, which is comparable with that reported previously
[19]. As expected, the PIP-I sequence without C646 did not possess HAT inhibitory activity.
Concomitantly, all C646-PIP derivatives, regardless of linker region and PIP sequence, maintained
HAT inhibitory activity, with IC50 values around 3.9–4.2 µM. These data verify that C646-PIP
preserved p300 inhibitory activity and the changes in PIP sequence had no obvious deleterious
effects on IC50. In addition, the inhibitory activity of C646-PIP was slightly decreased compared
with the C646 monomer, which might be attributed to the constraints exhibited by PIP moiety over
C646 binding to HAT.
2.4 Antiproliferative activity assay
Previously, antiproliferative activity of HAT inhibitors was shown in cancer cell lines [3, 20].
After a 10-day treatment, C646 exhibited an IC50 of 10.0, 6.0, and 6.3 µM in A549, H157, and
H460 cells, respectively, as assessed by a clonogenic survival assay based on a published article
[21]. Under ionizing radiation therapy, C646 radiosensitized lung cancer cells by enhancing the
mitotic catastrophe. However, only a limited dose ratio enhancement of 1.2–1.4 fold was observed
in this study to suggest the need to incorporate an alternative molecular design.
Figure 3. Cell viability assay in the A549 cell line using the WST-8 method. A549 cells were grown in 10%
FBS/DMEM medium for 24 h. Subsequently, the medium was replenished with varying concentrations of C646-PIP
conjugates (0.1, 1, 5, and 10 µM) for 96 h. The cell viability of A549 cells was measured using the WST-8 method.
Each bar represents the mean ± SD from three wells. P<0.05.

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Here, we studied the antiproliferative activity of C646-PIP using a cell viability assay in A549 cells
[22]. In consistent with our notion, A549 cell viability was not affected by PIP-I and C646, even at
the concentration of 10 µM, after 96 h of treatment (Figure 3). Treatment with C646-PIP
conjugates led to cell inhibition, but the extent of activity varied considerably among conjugates
with different linker lengths and PIP sequences.
Regarding the shared PIP-I sequence, 2 (moderate linker with a 13-atom distance) exhibited the
highest antiproliferative activity, with an IC50 of 1.8 µM, in a dose-dependent manner. This implies
that the length of the linker plays a pivotal role regarding its cellular effect, which was different
from the simplified HAT inhibitory activity in vitro. If the linker length was too short (1), C646
may not be sufficiently free to bind to the active pocket of p300 while PIP bound to DNA. In
contrast, a linker that was too long (3) may push C646 out of the localized region of PIP binding
thereby hampering the sequence-specificity. 2 and 4 had the same linker length (13-atom distance),
but different PIP sequences. However, 4 showed only a slight antiproliferative activity in A549
cells to imply that the PIP-I sequence is necessary for antiproliferative activity.
2.5 Microarray analysis
Microarray analysis has been used widely to identify underlying regulatory gene patterns [23].
In this work, whole-genome transcriptional profiling of the A549 cell line was performed using
microarray analysis, according to the manufacturer’s protocol [24]. 2 (2 µM) and 4 (2 µM) were
added respectively, for 48 h. 4 did not exhibit a specific gene regulatory pattern, which was
consistent with its low antiproliferative activity. After treatment with 2, among the more than
50,000 transcripts identified, 310 target genes showed 1.5-fold changes in expression levels
(P<0.05), with upregulation observed for 170 transcripts and downregulation detected for 140
transcripts. Genes detail information of microarray data up- and downregulated by treatment with 2
with levels >1.8-fold and <–1.8-fold changes (p<0.05) by Affymetrix gene expression console
software (Table 2).
Table 2. Genes detail information of microarray data up- and downregulated by treatment with 2. Genes
with >1.8-fold and <–1.8-fold changes (P<0.05). Uncharacterized transcripts were excluded from this table. Gene
symbols in red color and green color are upregulated and downregulated p53 downstream targets, respectively.
Gene Symbol Fold Change Gene Symbol Fold Change
SPATA18
EDA2R
ABCA12
BTG2
MIR3189
CDKN1A
SULF2
WDR63
FDXR
MDM2
UIMC1
MIR4713
SESN1
SCARNA7
MIR3685
CYFIP2
POLH
2.98
2.53
2.42
2.35
2.32
2.28
2.13
2.03
2.00
1.97
1.94
1.94
1.91
1.89
1.89
1.88
1.88
TP53INP1
1.85
CD70
1.83
FAS
1.83
GDF15
GAS6-AS1
ANKRD1
DDB2
1.81
1.81
1.81
1.80
NEFL
1.80
SNORA9
ALDH1L2
CARS
-1.81
-1.86
-1.90
-2.01
-2.01
-2.34
-2.34
-2.63
-3.64
ASNS
TAS2R30
STC2
ULBP1
GUSBP3
CHAC1
We performed an Ingenuity Pathway Analysis (IPA) of the differentially expressed genes, to gain a
more precise global understanding of the underlying biological processes (Figure 4a) [25].

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Interestingly, while 4 did not show potent and specific gene pattern regulations, treatment with 2
led to a strong enrichment of p53 target genes, such as SPATA18 (2.98 fold), BTG2 (2.35 fold),
FDXR (2.00 fold), and MDM2 (1.97 fold), among the top regulated transcripts. We then performed
a Gene Set Enrichment Analysis (GSEA) of the significantly differentially regulated genes using
the published gene expression profiles of p53 targets with gene sets of the Kyoto Encyclopedia of
Genes and Genomes (KEGG) (Supplementary Data S5) [26]. Consistent with the IPA analysis,
GSEA demonstrated that the p53 signaling pathway was predominantly enriched among all 185
gene sets identified, with an enrichment score of 0.67 and a P value <0.001(Figure 4b).
Figure 4. a) Significantly enriched target genes annotation in p53 by IPA. Targeting genes depicting the effect of 2
(compared with DMSO) the expression pattern of p53 pathway target genes in the technical duplicates of whole
transcriptome analysis (P<0.05) regulated at least 1.8-fold. b) A Gene Set Enrichment Analysis (GSEA) was
performed to assess whether genes that were differentially repressed by 2 were significantly associated with p53
direct effectors expression profiles of KEGG gene sets and gene symbols (GSEA database
c2.cp.kegg.v5.2.symbols.gmt). The enrichment score clearly showed that the genes at the top of the ranked list
were overrepresented in the reference gene set. P<0.001 and q<0.001.
A549 cells contain wild-type p53. Previous reports demonstrated that wild-type p53 plays a
significant role in tumor inhibition. Stabilizing p53 and selectively activating p53 targets in p53
wild-type cells resulted in a pronounced antiproliferative effect by inducing cell-cycle arrest and
apoptosis [27]. SAHA-I and CTB-I activated SOX2, OCT4, and NANOG in somatic human cells,
while showing no antiproliferative activity, and treatment with 2 showed slight decreases of
pluripotent genes after detail inspection of microarray data. Previous report demonstrated that
downregulation of p53 pathway is required for efficient iPS cell reprogramming and maintenance
[28]. Our data suggest that the inhibition of pluripotent genes might result in p53 pathway
activation based on the microarray data. Furthermore, 2 showed weak antiproliferative activity in
p53-mutant SW620 cells. Therefore, these results combined with the cell viability assay and
microarray experiment discussed above led us to conclude that the antiproliferative activity caused
by 2 in A549 cells was mainly attributable to the activation of p53 targeting genes.
2.6 Apoptotic assay
Among the many roles of tumor suppressor p53, the role of p53-dependent apoptosis in
pathological conditions was identified and well-documented, especially in cancer [27]. The

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microarray results led us to presume that p53-denpendent apoptosis might be the antiproliferative
mechanism underlying the effects observed for 2.
To confirm this hypothesis, cellular apoptosis was investigated via Annexin V-FITC assay, together
with living cells and dead cells [29]. A549 cells were treated with 2 and 4 for 48 h. Subsequently,
apoptotic, necrotic, and healthy cells were stained with three different fluorophores (Annexin
V-FITC, ethidium homodimer III, and Hoechst 33342) (Figure 5).
Figure 5. Effect of 2 and 4 on cellular apoptosis and necrotic activity. Representative images of apoptosis
detection using fluorescence imaging. A549 cells were treated with DMSO, 2, and 4 at 2 µM for 48 s. A confocal
image showed Hoechst 33342 staining, Annexin V-FITC staining, ethidium homodimer III staining, and an overlay
of the images. (Scale bars represent 100 µm).
In accordance with the IC50 of 2, 2 µM 2 and 4 were applied to observe the process of cell death. In
the control experiment (0.1% DMSO), apoptotic/dead cells were difficult to detect. Conversely,
cultures treated with 2 showed more abundant apoptotic cells and an obvious subset of dead cells,
which was consistent with the results of the cell viability assay described above. For 4, most of the
cells were in a healthy condition, and only a few necrotic cells and apoptotic cells were detected. As
wild-type p53 plays a major role in A549 cell apoptosis, it is reasonable to assume that 2 activated
p53 downstream targets and induced cell apoptosis, leading to programmed cell death.
To validate the role of p53 in antiproliferative and apoptotic activities of 2, we further tested 2 in
wild-type p53 cell line (MV4-11 cells) and mutant p53 cell lines (SW620 and MDA-MB-231 cells)
for 96 h (Supplementary Data S6). Consistent with antiproliferative activity in A549 cells, 2
showed IC50 of 2.6 µM in MV4-11 cells. On the other hand, results showed that 2 had weak
inhibitory activity in mutant p53 cell lines SW620 cells (78.4% cell viability ) and MDA-MB-231
cells (69.3% cell viability) at the concentration of 10 µM. Above all, the antiproliferative and
apoptotic activity of 2 might mainly mediated by wild-type p53 rather than mutant-p53.

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3. Conclusions
In addition to DNA mutation, heritable phenotypes, such as the inactivation of tumor suppressor
genes, can also be acquired by epigenetic modifications, including DNA methylation and histone
modifications [30]. Inactivation of tumor suppressors and their downstream pathways, such as the
p53 pathway, or activation of proto-oncogenes and their correspondent pathways, such as the RAS
pathway, have been extensively studied to play a pivotal role in tumor initiation and development.
In particular, wild-type p53 is popularly known as the guardian of the genome owing to its
significant role in tumor inhibition. Stabilizing p53 and selectively activating the p53 pathway in
p53 wild-type cells resulted in a pronounced antiproliferative effects via the induction of cell-cycle
arrest and apoptosis [31]. In contrast, mutations in the p53 tumor suppressor gene are associated
clinically with cancer stem cell progression and metastasis [32, 33].
In this study, we report for the first time about the synthesis and characterization of a new type of
dual-functional conjugates comprised of a selective DNA-recognizing small molecule and a
synthetic inhibitor of HAT activity. Our predicted working model about dual functionality, i.e.,
sequence-specific DNA binding and HAT inhibitory activity got substantiated using thermal
stability assay and HAT activity assay, respectively. To evaluate the bioactivity of our
dual-functional conjugates, we investigated the antiproliferative activities in p53 wild-type cell
lines (A549 and MV4-11 cells) and p53 mutant cell lines (SW620 and MDA-MB-231 cells).
Among C646-PIP derivatives synthesized, 2 (with a medium linker length of 13-atom) showed
potent antiproliferative activity with an IC50 of 1.8-2.6 µM in A549 and MV4-11 cells.
Additionally, the data acquired from microarray analysis and apoptosis assay, we demonstrated
that the antiproliferative mechanism of 2 occurred via the activation of p53 target genes, which in
turn triggered subsequent p53-dependent apoptosis. Potent antiproliferative activities and
multi-gene network regulation observed in this research on sequence-specific dual-target
epigenetic repressor offer us an alternative way to study and develop novel antitumor therapeutics.
4. Experimental section
4.1 General methods
Reagents and solvents were purchased from standard suppliers and used without further
purification. HAT inhibitor C646 was purchased from Sigma-Aldrich (SML0002-25 mg). HPLC
analysis were performed with a JASCO PU-2080 plus HPLC pump, a JASCO 807-IT HPLC
UV/Vis detector, and a Chemcobond 5-ODS-H reversed-phase column (4.6×150 mm) in 0.1ꢀ%
TFA in water using acetonitrile as the eluent at a flow rate of 1.0 mLꢀmin−1 under detection at 254
nm. Electrospray ionization Time-of-flight mass spectrometry (ESI-TOF MS) was performed on a
Bio-TOF II (Bruker Daltonics) mass spectrometer by using positive ionization mode.
Machine-assisted PIP syntheses were performed on a PSSM-8 (Shimadzu) system with
computer-assisted operation on a 20 µmol scale using Fmoc chemistry.
4.2 HAT inhibitor-PIP synthesis
Machine-assisted automatic solid-phase synthesis of hairpin-type PIP was carried out with the
use of a continuous-flow peptide synthesizer (PSSM-8, Shimadzu, Kyoto, Japan) at 0.1 mmol
scale (100 mg of Fmoc oxime resin, 0.50 meq/g, Peptide Institute, Osaka, Japan) [15]. In a
stepwise
reaction,
Fmoc-Py-COOH,
Fmoc-Im-COOH,
Fmoc-Py–Im-COOH,
Fmoc-β-alanine-COOH, 4-(Fmoc-amino) butyric acid and Fmoc-4,7-dioxanonanoic acid were

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coupled sequentially. Automatic solid-phase synthesis was started by washing with
dimethylformamide (DMF); coupling with a monomer for 60 min in an environment of
1-[bis(dimethylamino)methylene]- 5-chlorobenzotriazolium 3-oxide hexafluorophosphate
(HCTU) and diisopropylethylamine (4 eq. each); removing the Fmoc group using 20%
piperidine/DMF. We then isolated PIP after cleavage step (1 ml N,N-Dimethylpropylamine)
for compounds 1-4.
Through 11-step solid phase synthesis and reverse-phase flash column, four PIPs were
successfully synthesized. Then, our target compounds were synthesized and purified in high
yield by conjugating commercially available C646 with 1-NH2, 2-NH2, 3-NH2, and 4-NH2
respectively with the help of coupling agent PyBOP. To purify the PIP conjugates, high
performance liquid chromatography (HPLC) was performed with use of a PU-980 HPLC
pump, UV-975 HPLC UV/VIS detector (Jasco, Easton, MD), and Chemcobound 5-ODS-H
column (Chemco Scientific, Osaka, Japan). ESI-MS data are listed in Supplementary Data
S7-S11.
2+
PIP-I was obtained as a white powder. MS (ESI-TOF): m/z calcd for C57H71N21O10 [M+2H]2+:
604.7841; found: 604.7302; HPLC: tR=16.09 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
40 min).
2+
1-NH2 was obtained as a white powder. MS (ESI-TOF): m/z calcd for C61H77N23O11 [M+2H]2+:
654.8159; found: 654.6877; HPLC: tR=10.06 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
2-NH2 was obtained as a white powder. MS (ESI-TOF): m/z calcd for C64H85N23O13 [M+2H]2+:
691.8343; found: 691.6911; HPLC: tR=10.11 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
3-NH2 was obtained as a white powder. MS (ESI-TOF): m/z calcd for C67H91N23O15 [M+2H]2+:
728.8527; found: 728.7889; HPLC: tR=10.18 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
4-NH2 was obtained as a white powder. MS (ESI-TOF): m/z calcd for C63H84N24O13 [M+2H]2+:
692.3320; found: 692.3352; HPLC: tR=9.692 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
1 was obtained as a red powder. MS (ESI-TOF): m/z calcd for C85H96N26O16 [M+2H]2+:
868.3744; found: 868.2229; HPLC: tR=14.05 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
2 was obtained as a red powder. MS (ESI-TOF): m/z calcd for C88H102N26O18 [M+2H]2+:
905.3927; found: 905.3164; HPLC: tR=14.10 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
3 was obtained as a red powder. MS (ESI-TOF): m/z calcd for C91H108N26O20 [M+2H]2+:
942.4111; found: 942.4408; HPLC: tR=14.19 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
2+
4 was obtained as a red powder. MS (ESI-TOF): m/z calcd for C87H101N27O18 [M+2H]2+:
905.8904; found: 906.0553; HPLC: tR=13.83 min (0.1ꢀ% TFA/MeCN, linear gradient 0–100ꢀ%, 0–
20 min).
Polyamide concentrations were calculated with a Nanodrop ND-1000 spectrophotometer
(Thermo Fisher Scientific Inc.) using an extinction coefficient of 9900 M1 cm-1 per one
pyrrole or imidazole moiety at λmax near 310 nm [16]. C646-PIP concentrations were

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calibrated by comparing UV absorbance at 410 nm to C646 [13].
4.3 Thermal stability assay
Thermal stabilization of polyamide–DNA complex can be analyzed by thermal melting
temperature (Tm) analysis, and this method has been used for the measurement of the relative
binding affinity and the ability to discriminate mismatch sequences. Melting temperature
analyses were performed on a spectrophotometer V-650 (JASCO) equipped with a
thermos-controlled PAC-743R cell changer (JASCO), a refrigerated, and heating circulator
F25-ED (Julabo). The sequences of dsDNA were ODN1: 5'-ACTTATTCCATATAGA-3',
ODN2: 3'-TGAATAAGGTATATCT-5', ODN3: 5'-ACTTATCCACTATAGA-3', and ODN4:
3'-TGAATAGGTGATATCT-5'. The underlined bases were the binding sites of polyamides.
The analysis buffer was the aqueous solution of 10 mM NaCl and 10 mM sodium cacodylate
at pH 7.0 containing 0.25 % v/v DMF. The concentration of polyamides and dsDNA was 2.5
µM, respectively. Before analyses, samples were annealed from 95 ℃to 20 ℃ at a rate of
1.0 ℃/min. Denaturation profiles were recorded at λ= 260 nm from 20 to 95 ℃ at a rate of
1.0 ℃/min.
4.4 HAT activity assay in vitro
HAT activity assays were determined using a commercial available kit (Biovision
Biotechnology) according to the manufacturer’s instruction [12]. Biovision’s HAT Activity
Assay Kit utilizes Acetyl CoA and H3 histone peptide as substrates, and recombinant p300
core domain as the enzyme. In this assay, HAT enzyme catalyzes the transfer of acetyl groups
from Acetyl-CoA to the histone peptide, thereby generating two products - acetylated peptide
and CoA-SH. The CoA-SH reacts with the developer to generate a product that is detected
fluorometrically at Ex/Em = 535/587 nm.
4.5 Cell culture
To make the complete growth medium for A549 cell line: ATCC-formulated DMEM medium,
fetal bovine serum (Sigma FBS) to a final concentration of 10% and L-glutamine to a final
percentage of 2 mM. To make the complete growth medium for MV4-11 cell line: RPMI
1640 medium, FBS to a final concentration of 10%, L-glutamine to a final percentage of 2
Mm and penicillin/streptomycin to a final concentration of 1%. To make the complete
growth medium for SW620 cell line, we added the following components to the base medium:
ATCC-formulated DMEM medium, FBS to a final concentration of 10% and
penicillin/streptomycin to a final concentration of 1%. To make the complete growth medium
for SW620 cell line, we added the following components to the base medium:
ATCC-formulated DMEM medium, FBS to a final concentration of 10% and
penicillin/streptomycin to a final concentration of 1%. To make the complete growth medium
for MDA-MB-231 cell line, we added the following components to the base medium:
ATCC-formulated DMEM medium, FBS to a final concentration of 10% and
penicillin/streptomycin to a final concentration of 1%.
4.6 Cell proliferation assay
Cancer cell lines were seeded on 96-well microplates (5×103 cells/well) in above mentioned

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media for 24 h at 37oC in 5% CO2. Then media was replenished with varying concentrations
of conjugates C646-PIP for 96 h. Count Reagent SF (Nacalai Tesque) was added to each well
to evaluate the cell proliferation. The absorbance of each well was measured at 450 nm by
SpectraMax M2 (Molecular devices) microplate reader.
4.7 Microarray analysis and data processing
A549 cells were plated in 6-well plates at 2 × 105 cells/well and were treated for 48 h with
DMSO (0.01% control) and 2 µM concentration of C646-PIP, with technical duplicates in
each condition respectively. Total RNA was prepared and the integrity of the RNA was
checked using the Agilent 2100 Bioanalyzer (Agilent Technologies). One hundred nanograms
of total RNA quantified by Nanodrop ND1000 v. 3.5.2 (Thermo Scientific) was labeled using
a GeneChip WT PLUS Reagent kit (Affymetrix) and was hybridized to Human Gene 2.1 ST
Array Strip (Affymetrix) for 20 ± 1 h at 48°C. The hybridized arrays were washed, stained,
and imaged on a GeneAtlas Personal Microarray System (Affymetrix). The hybridized probe
set values were normalized using Affymetrix gene expression console software. Significant
differentially expressed genes between the different conditions were analyzed using t test
statistics (p<0.05). Microarray data reported here were deposited in the Gene Expression
Omnibus database under the accession number (currently unavailable).
Gene set enrichment analysis (GSEA) was conducted to detect statistically significant 2
targeted genes associated with a gene set p53-regulated genes. Data were further analyzed by
IPA (Ingenuity Systems; http://www.ingenuity.com) [26]. The IPA functional analysis was
conducted to identify the significant biological functions associated with the microarray data
set. Genes from the data sets that were regulated by ≥1.8-fold cutoff at p<0.05 were
considered for the analysis. The p value was calculated using right-tailed Fisher’s exact test
that defines the degree of association of the data set to the assigned biological function.
4.8 Apoptosis Detection assay
The apoptosis detection assay was performed using an Apoptotic, Necrotic, and Healthy Cells
Detection Kit (Promokine) according to manufacturer’s instructions [29]. A549 cells were
cultured in 8-well chamber slides at a density of 2×104 cells/well and treated with 2 µM PIP
for 48 hr. Cells were washed with a binding buffer and stained with FITC-Annexin V,
Ethidium Homodimer III, and Hoechst 33342; then they were subjected to fluorescence
microscopy analysis (SpectraMax M2, Molecular Devices).
4.8 Statistical analysis
Results for continuous variables were presented as the mean ± standard error. Two-group
differences in continuous variables were assessed by the unpaired T-test. P-values are
two-tailed with confidence intervals of 95%. Statistical analysis was performed by comparing
treated samples with untreated controls.
Acknowledgments
This work was supported by JSPS KAKENHI Grant NO. JP16H06356, “Basic Science and
Platform Technology Program for Innovative Biological Medicine by Japan Agency for Medical
Research and Development (AMED)”, “the Platform Project for Supporting Drug Discovery and

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Life Science Research funded by AMED” and “JSPS-NSF International Collaborations in
Chemistry (ICC)” to G.N.P., Grant NO. 16K12896. We also thank China Scholarship Council
(CSC) support Z. Y., Japanese Government (MONBUKAGAKUSHO: MEXT) Scholarship support
Y. W., and JSPS scholarship support J. T.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/.
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Graphical abstract
Highlights
 Sequence-specific HAT inhibitors C646-PIP were reported here for the first time and predicted
working assay, including thermal stability assay and in vitro HAT activity assay, showed these
conjugates retained dual-targeting activities;
 In p53 wild-type cancer cell lines, 2 which contains medium linker length of 13-atom and
PIP-I sequence was identified with highest antiproliferative activity with an IC50 of 1.8-2.6
M in A549 and MV4-11 cells;
 Highly regulated p53 downstream targets and cancer cell apoptosis contribute to
antiproliferative activity of 2.