Applications of PIP-Pyrrole-Imidazole PolyamideLiterature seminar #1
2024/1/25
B4 Mayo YamazakiContents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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2

Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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3

What is PIP (pyrrole-imidazole polyamide)?
Introduction

ü PIP is a programmable DNA minor groove binder.
ü PIP selectively recognizes the four base pairs in duplex DNA.
l Im/Py pair → G/C base pairl Py/Py pair → A/T or T/A base pair
ü Kd < 1 nM (similar to DNA-binding proteins)
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Trauger, J. W., Dervan, P. B. et al. Nature 1996, 382, 559–561

Dervan, P. B. & Edelson, B. S. Curr. Opin. Struct. Biol. 2003, 13, 284-299

4A Brief History of PIP
Introduction

Natural antibiotics

Pyrroleü Netropsin binds to A/T sequences in the DNA minor groove by forming a 1:1 complex. (1985)
Dickerson, R. E. et al. J. Mol. Biol. 1985, 183, 553–563

ü Distamycin A binds to A/T sequences in the DNA minor groove by forming 2:1 complex. (1989)
Wemmer, D. E. et al. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5723–5727

ü Replacing Py with Im enabled the recognition of G/C sequences.

Dickerson, R. E. et al. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376–1380
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Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85
Trauger, J. W., Dervan, P. B. et al. Nature 1996, 382, 559–561 Dervan, P. B. & Edelson, B. S. Curr. Opin. Struct. Biol. 2003, 13, 284-299

5Applications of PIPsex) Single PIP can inhibit transcription factor

Variety of applications of PIP

Introduction

Today’s topic

Ø Designing PIP to bind to SOX2 target sequenceØ The PIP inhibited SOX2 and promoted the transcriptionof Mesodermal genes
Taniguchi, J., Sugiyama, H. et al. Nucleic Acids Res. 2017, 45, 9219–9228
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Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85

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Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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A novel approach directly targeting the mutant DNA
KR12

lOncogenic driver mutations = Target for therapyex) KRAS mutationlMany colon cancer patients have the KRAS (G12D)
mutation or the (G12V) mutation.
lDirect pharmacological targeting of activated KRAS
has not led to clinical application.

This work
new approach

Directly targetingmutant RAS genes

ü Development of PIP-indole-seco-CBI conjugatestargeting KRAS codon 12 mutations
Drug resistance
Undesirable
cell growth

CellgrowthTakashima, A. et al. Expert Opin. Ther. Targets. 2013, 17, 507-531

Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85 2024/1/25

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KR12 targets KRAS codon 12 (G12D and G12V) mutants
KR12

G12D
Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85

G12V
Summary table of the dissociation constants

ü KR12 preferentially bound to the mutant
KRAS sequences compared to the wild type.
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Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

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KR12 alkylated at mutated KRAS codon 12 sites
KR12

l Thermally induced strand cleavage procedureü Dose-dependent alkylation by KR12 was detected
only at the mutated KRAS codon 12 target sites
DNA alkylation was visualized by thermalcleavage of the 5ʹ-Texas Red labeled DNA
strands at the alkylated sites, which displayedcleavage bands quantitatively on thepolyacrylamide gel.
5ʹ-Texas Red-labelled DNA fragments
containing the indicated KRAS codon 12
mutations were incubated with the indicatedconcentrations of KR12 for 10 h at room
temperature, followed by the addition of calf
thymus DNA. The reaction mixtures werethen incubated at 90 °C for 5 min to cleave
DNA strands at their specific alkylated sites.
DNA fragments were then recovered by
vacuum centrifugation, dissolved in loading
dye, denatured at 95 °C for 20 min andsubjected to electrophoresis on a 6%
denaturing polyacrylamide gel.

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KR12 inhibited cell growth of KRAS mutated human cancer cell
KR12

human colon cancer cell lines

WT

G12C

G12V or G12D

Target of KR12l Analysis of cell growth inhibition by KR12

Figure 2 | KR12-mediated specific suppression of KRAS codon 12 mutants in human colon cancer
cells. (a) WST assay. Human colon cancer cells expressing wild-type KRAS, including HT29 andCaco-2 or human colon cancer cells harbouring KRAS codon 12 mutations, including SW1643,
SW480, SW620 and LS180, were incubated with the indicated concentrations of KR12. 48 hours
after treatment, the percent viable cells were examined by WST assay and depicted in a line
graph. Error bars indicate the s.d. of the data from triplicate experiments.

Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

After KR12 treatment
ü Viability of KRAS G12D or G12V mutant
cells was significantly lower

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KR12 induced mutant KRAS suppression
KR12

Analysis of the specific alkylation and subsequent downregulation after KR12 treatment
Human colon cancer-derived, heterozygously mutated LS180 (12D/WT) cells#6 = not bound to the codon 12 mutant KRAS sequences

In Cell
Endogenous alkylation confirmed by
Ligation-mediated PCRKRAS mRNAKRAS protein

active GTPbound form

ü KR12 induced DNA
alkylation in LS180 cells.

KR12 lacking
seco-CBIWT(b) Quantitative reverse transcription-PCR analysis. Relative KRAS expression 48 h after treatment with DMSO, KR12, #6 or Dp was plotted as a bar
graph. Error bars indicate the s.d. of data from triplicate experiments. (d) Immunoblot analysis. Immunoblots for anti-KRAS or anti-actin antibody(top and bottom panels, respectively) for LS180 (12D/WT) and HT29 (WT) cells 48 h after the treatment with either control DMSO solution, KR12 or#6. The GST-Raf-bound proteins from each treated group were pulled down and analysed by immunoblotting with anti-RAS antibody (middle panels).

ü Significant downregulation of total KRAS RNAü Specific reductions in activated KRAS protein levels in LS180 cells
Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

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KR12 showed tumor growth reduction in vivo
KR12

(WT)

Body weight (g)White = Control (DMSO)Black = KR12-treated group(12D/WT)(12V/12V)

Figure 4 | KR12 suppresses tumour growth in vivo. (a) The indicated human colon cancer
cells, that is, HT29 (WT), LS180 (12D/WT) and SW480 (12V/ 12V), were injectedsubcutaneously into BALB/c nude mice. When the tumour volume reached 100 mm3, DMSOor KR12 (320 mg kg 1 body weight) was intravenously injected through the tail vein every 7
days. At the indicated times after administration, the tumour volume was calculated as 1/6
p the longest diameter width height. The mean tumour volume, with s.e. (open circles for
DMSO and closed circles for KR12 treatment), and mean body weight, with s.e. (opentriangles for DMSO and closed triangles for KR12 treatment), of the KR12-treated group andcontrol group are plotted in a line graph with error bars (s.e.). The numbers of animals usedwere 6 for HT29 (DMSO and KR12) and LS180 (KR12), 5 for LS180 (DMSO) and SW480(KR12), and 8 for SW480 (DMSO). (b) Images of the euthanized mice of each group areshown. An image of an SW480 xenograft 5 weeks after KR12 treatment, which was the final
treatment, is also shown.

KR12-treated LS180 and SW480
ü Significant reduction of tumor growthwith minimum toxicity
Tumour volume (103 mm3)
Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

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KR12 showed lower toxicity than CBI without PIP
In Cells

cell lines with KRAS mutations
recognized by KR12

In vivoKR12

l CBI itself treatment
Ø In cells
IC50 did not change depending on KRAS codon 12 mutant.
Ø In vivo
Tumour growth reduction was slightly weaker than KR12 treatment.
Mice showed weight loss and slight illness.
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Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

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Summary of KR12 & Challenges
SummaryKR12…
üalkylated mutant DNA sequence selectively.
üdownregulated active KRAS.
üsuppressed the growth of KRAS G12V/G12D mutant tumor
in vivo.
üreduced the toxicity of the alkylating agent.
üproduced effects that were not achieved by PIP alone or
the DNA alkylating agent alone.
ChallengesØOptimization to improve their specificity
Ø9,121 target sites = Potential off-target of KR12
Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

KR12

pyrrole–imidazole polyamide
indole-seco-CBI conjugate

Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85
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Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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Acetylation of histone lysine residues regulates transcription
Bi-PIP

Acetylation of histone lysine residues is strongly correlated with transcriptional activation.
lopening the chromatin structure
lrecruiting proteins containing bromodomain (BD)
NH2
NH2

NH2

NH2

NH2

Histone acetyl transferase (HAT)

O
NH

NH2

O
NH

Histone deacetylase (HDAC)

Repressed

repress transcription
gene
Taniguchi, J., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 7108–7115

Activepromote transcriptiongene2023/11/07

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Previous design to achieve sequence-selective histone acetylation
Bi-PIP~45-fold~20000-fold

SAHA-PIP

RecentØ fusion of P300 (containing HAT) withCRISPR/Cas system×need the transfection-based system×enzymatic degradation in cell
Hilton, I., Gersbach, C. et al. Nat. Biotechnol. 2015, 33, 510–517

SAHA-PIP

Previous effort using PIP
Ø SAHA (HDAC inhibitor) –PIP
→ inconsistent of the level of gene activation because ofindirect manner
Pandian, GN., Sugiyama, H. et al. Sci. Rep. 2014, 4, 3843

Yamatsugu, Kawashima, Kanai Curr. Opin. Chem. Biol. 2018, 46, 10–17
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Bi-PIP: Conjugates of a BD inhibitor (Bi) and PIP
Bi-PIPl Some proteins have both HAT domain
and bromodomain (BD).
l One acetyl mark causes acetylation ofneighboring sites via BD-mediatedrecruitment of these proteins.

This work
Localization to a specific DNA sequence of a BD inhibitor by “Bi-PIP”→ Localization of a HAT-BD protein → Locus-specific histone acetylation

Figure 1. Strategy of targeted histone
acetylation utilizing the bromodomain(BD)-mediated propagation ofacetylation. (a) Structure of P300/CBP
coactivator proteins. NRID, nuclear
receptor interacting domain; TAZ1,transcriptional adaptor zinc finger 1;
KIX, KID-interacting; BD, bromodomain;
RING, really interesting new gene; PHD,
plant homeodomain; HAT, histone
acetyltransferase; ZZ, ZZ-type zincfinger; TAZ2, transcriptional adaptor
zinc finger 2; IBiD, interferon-binding
domain. (b) BD-mediated propagationof histone acetylation by P300/CBP.
Existing acetyl lysine (yellow) is
recognized by BD of P300/CBP, and denovo acetyl modification (green) is
introduced by HAT domain. (c) Designof artificial epigenetic code ofacetylation named “Bi-PIP” (left) andmodel of targeted histone acetylation
by Bi-PIP (right). Bi mimics acetyl lysine
to recruit P300/CBP through its BD.

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Design of Bi-PIP
l Bi = one of the CBP30 (P300/CBP selective BD inhibitor) derivativesl The target sequence of PIP
Bi-PIP1 → 5’-WWCWGWCW-3’ (8bp)
Bi-PIP2 → 5’-WWCCGCCW-3’ (8bp)(W = A or T)

Bi-PIP

Figure 2. Chemical structures of Bi (1), Bi-PIP1 conjugate (2), PIP1 monomer (3),Bi-PIP2 conjugate (4), and PIP2 monomer (5).

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Bi-PIPs promote histone acetylation on nucleosomes that have theirtarget DNA sequences in vitro
Bi-PIPc

Figure 3. Bi-PIPs promote histone acetylation on
nucleosomes that possess their target DNA
sequences. (c) HAT reaction–in vitro ChIP-qPCR
was performed with a series of P300
concentrations (0, 3, 10, 30, 100 nM) with orwithout 100 nM Bi-PIP1. Positive control
represents a nucleosome with synthetic acetylated
histone H3 (K4ac, K9ac, K14ac, K18ac, K23ac). (d)HAT reaction–in vitro ChIP-qPCR was performed
with 10 nM of P300. Each compound was applied
at a concentration of 100 nM. (e) HAT reaction–invitro ChIP-qPCR was performed Bi-PIP1 (100 nM)
or Bi-PIP2 (100 nM) with 10 nM of P300.

ü 100 nM Bi-PIP1 induced intensive acetylation on its target Nuc1 but
not on Nuc2.
ü The level of acetylation was dependent on the concentration of P300.

ü Acetylation was achieved by PIP binding to the target DNA
sequence selectively and recruiting P300 by the BD-Bi interaction.
Taniguchi, J., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 7108–7115

Bi alone, PIP alone,
and mixtures thereof
do not promote
acetylation.

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Cell permeability of PIPs
Bi-PIPFITC

HEK293T cells
without Endo-Porter

with Endo-Porterl The cell permeability of PIPs = vary dependingon the chemical structures and cell linesü Endo-Porter was used to ensure that all of thecompounds enter the cells.

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Bi-PIP caused greater changes in gene expression
Bi

PIP1

Bi-PIP1

Bi-PIP2

PIP2

Bi-PIP

HEK293T cells
A transcriptome analysis of total RNA
extracted from cells

ü Bi-PIP1 and Bi-PIP2 gave
greater transcriptome changes,
mainly for activation.
Fig.S10. Genome-wide gene expression change by each compound.
(a–e) Scatter plots of cells treated with Bi (a), PIP1 (b), PIP2 (c), Bi-PIP1 (d),or Bi-PIP2 (e) versus vehicle-treated cell. Red and green dots represent
transcripts which were up- or downregulated in each comparison. >2 or <−2fold change and <0.05 P value were used as the criteria. (f) Summary of thenumber of differentially expressed genes.
After 15 hours of compound treatment, RNA was extracted. 2024/1/25

Taniguchi, J., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 7108–7115

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Bi-PIPs selectively activated gene expression in living cells
Bi-PIP

Uniquely activated transcripts by Bi-PIP and Bi-PIP2→ NTS (neurotensin) and IER5L (immediate early response 5-like)l NTS locus contains the binding sites for Bi-PIP1 but not for Bi-PIP2.
l IER5L locus contains the binding sites for Bi-PIP2 but not for Bi-PIP1.

Figure 4. Bi-PIPs epigenetically activate selective gene expression inside living cells. (b) Volcano plots of transcriptome comparison of Bi-PIP1 vs vehicle (left) and Bi-PIP2 vs vehicle (right). Uniquely upregulatedtranscripts by individual Bi-PIP (>1.5 fold change) are indicated as colored dots. (c) Expression of NTS and IER5L was confirmed by RT-qPCR. Error bars represent standard deviation of data from two culture wells.
2024/1/25(d) Genomic loci of NTS (top) and IER5L (bottom). Match sites for Bi-PIP1 (5ʹ-WWCWGWCW-3ʹ) and Bi-PIP2 (5ʹ-WGCCGCCW-3ʹ) are indicated as blue and red diamonds, respectively.

Taniguchi, J., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 7108–7115

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Acetylated loci by Bi-PIPs
Bi-PIPü The acetylation at NTS locus slightly increased
by Bi-PIP1.
ü The acetylation at IER5L locus increased by Bi-PIP2.

anti–acetylated H3△ Acetylated loci by Bi-PIP= its target sequences + the other possible binding sites
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Summary of Bi-PIP & Challenges
Bi-PIP

Summaryü Bi-PIP recruited P300/CBP to selective DNA sequences.
ü Bi-PIP promoted the sequence-selective histone acetylation and transcriptional activationin living cells without gene transfection.
Challenges
Ø Need to improve the sequence-selective ability
Ø Endogenous enzyme-dependent

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Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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27

TFs often act as homo/heterodimers
Current challenges
Ø The short recognition sequence of PIP leads high off-target rates.
Ø The extension of PIP length signiﬁcantly reduces its cell permeability.
Ø Single PIP cannot block interactions between TF pairs and DNA.

PIP-HoGu※ Transcription factors (TFs) often act ashomo/heterodimers→ high binding affinity andextended recognition sequences
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Yu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

Molecular Biology of the Cell 6th edition (2017)

F7-9,18

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Design for mimicking cooperative TF-pair systems
PIP-HoGu

Strategyl Integration of PIPs with a cooperative system→Noncovalent cooperative system(nucleic acid analogues, metal ion-ligand,host-guest systems (ex. cyclodextrin(Cyd)adamantane(Ada)))mini-PEG
linkerFigure 1. Overview of cooperative interactions of a TF pair targeting a sequence
associated with two components of Pip-HoGu assembly, Ada1 and Cyd1. n = gap
distance. (Bottom) Chemical structures of Ada1, Ada2, and Cyd1.

Yu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

This work
1. Design PIP-HoGu (PIPs conjugated to a host-guest
Cyd-Ada scaffold)
2. Evaluate PIP-HoGu in vitro using the DNA binding
sequences of the Tax/CREB heterodimer
3. Experiment in cells
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0-5 bp gap distances displayed cooperative binding.
impossible for Ada
to interact with Cyd

PIP-HoGu

Thermal stabilization assay(∆" = difference in melting temperature)ü improved thermal stability by thecooperative interaction of the Cyd–Ada
complexesü gap 0-5 bp → cooperative binding
functionü 2 bp gap → the highest level ofcooperation (ΔΔTm=7.2 ℃)
Figure 2. Tm assay illustrating the cooperativity of Pip-HoGu. (A) The DNA oligomers(ODNs) used in the Tm assay, including positive (ODN1ʹP−ODN6P) and negative(ODN1ʹN−ODN6N) binding sequences. The gap distance (green) is the number of bp
between the binding sites of Ada1 (blue) and Cyd1 (red). The chart only shows theforward DNA strand. (B) Tm proﬁles of positive ODNs (TmP, light blue), negative ODNs
(TmN, gray), positive ODNs/Ada1− Cyd1 (TmPA, blue), and negative ODNs/Ada1−Cyd1(TmNA, black). (C) ΔΔTm proﬁles of cooperativity of Ada1−Cyd1 assemblies. ΔTm = Tm
(ODNs/PIPs) − Tm (ODNs); ΔΔTm = ΔTmP − ΔTmN. Error bars indicate the standard
deviation of three replicates.
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PIP-HoGu has high sequence-selectivity.
cf)

PIP-HoGu

Single PIP
3-bp mismatchl 1-bp mismatch Tm assay
ü PIP-HoGu exhibited highsequence-selectivity.
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Yu, Z., Sugiyama, H. et al. Eur. J. Med. Chem. 2017, 138, 320−327

31PIP-HoGu inhibited natural TF-pair binding in cells.
PIP-HoGu
Figure 5. Cell-based assay of Pip-HoGu. (A) Crystal
structure of Erα homodimer and DNA sequence.
(B) Schematic diagram of ERE-driven luciferase in
T47D-KBluc cells. (C) The structural design of
Ada3 and Cyd2 targeting ERE sites (Figure S8). (D)Luciferase activity assay after normalization to
the total protein concentration.

※ERE (estrogen response element)= specific target motif ofthe estrogen receptor α (ERα) homodimer

moderate-to-weak inhibitory
activity (IC50≥2µM)
very weak activity(<20% inhibition)

4-5-foldenhancementof theinhibitory effectYu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

l cell-based assay
In ERα-positive,
17β-estradiol-stimulated T47D-KBluc cells(highly express luciferase after binding
three ERα TF pairs)
Ada3 only vs. Cyd2 only vs. Ada3-Cyd2→ luciferase activity was measured

ü PIP-HoGu potently inhibited
natural TF pairs in cell.
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Summary of PIP-HoGu & Challenges
Summary

PIP-HoGu

Challenges
Ø Limitation of gap distance (~5 bp)
Ø Optimization of the host-guest moiety
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Yu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85

33

Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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34

A synthetic mimic capable of cooperative DNA binding andepigenetic modulation

ePIP-HoGu

Illustration of ePip–HoGu system

Motivation
TFs → cooperative DNA binding & transcriptional modulation
Need to create a synthetic mimic that can perform both cooperative DNA binding andepigenetic modulation
Strategy
The installation of an epigenetic modulator (epi-drug) to PIP-HoGu
Yu, Z., Sugiyama, H. et al. Chem. Commun. 2020, 56, 2296

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Upgraded the cooperation domain in PIP–HoGu system
ePIP-HoGu

Improvement of PIP-HoGu replacing
Cyd with CB7*CB7 = cucurbit[7]urill CB7-PIP (Cuc1)l Ada1-6 (Linker length, type, andpositive charge are different)
Fig. 2 Chemical structures of host conjugates CB7–PIP and Cyd–PIP (A),and guest conjugates Ada–PIP (B).

Yu, Z., Sugiyama, H. et al. Chem. Commun. 2020, 56, 2296

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Cuc1 exhibited higher affinity than Cyd1
ePIP-HoGu

Thermal stabilization assay
ü Cuc1 exhibited higher thermal stability than Cyd1
ü Ada3 (with an ethyldiamino residue and alkyl chain) showed
the most prominent stabilization
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ePIP-HoGu recruited functional enzymes sequence-selectively.
ePIP-HoGu

ü Co-treatment of Ada_Bi1 and Cuc2 hugely increased theacetylation level in Nuc1 (nearly 20-fold)ü Co-treatment of Ada_Bi1 and Cuc_Bi1 further enhanced theacetylation level in Nuc1 (23.5-fold)
Yu, Z., Sugiyama, H. et al. Chem. Commun. 2020, 56, 2296

Fig. 4 ePIP–HoGu synergistically recruits an epigenetic modifier to the target DNA repeat locus.
(A) Schematic illustration of four kinds of nucleosomes with different DNA templates. Nuc1
contains four-matched repeat sequence of PIP–HoGu binding. Nuc2 has two homodimericbinding sites of Ada–PIP and CB7–PIP separately, which cannot form a host–guest interaction(Nuc2 has potential synergic binding partially between site 2 and 3, because of the shortdistance between them). One-mismatch bp localizes in the binding site of Ada-PIP for Nuc3
and CB7-PIP for Nuc4. (B) The workflow of the in vitro HAT-ChIP-qPCR assay. (C) Results of
the in vitro HAT-ChIP-qPCR assay. Compound treatment in three groups compared with
control (DMSO), i.e., Ada_Bi1, Ada_Bi1 + Cuc2, and Ada_Bi1 + Cuc_Bi1.
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Summary of ePIP-HoGu & Challenges
Summary
ü ePIP-HoGu contain a DNA binding domain,
an interaction domain, and a gene regulatory domain
like natural TF pairs.
ü ePIP-HoGu promoted histone acetylation selectivly.

ePIP-HoGu

Challenges
Ø Endogenous enzyme-dependentØ Cell-based assay
Ø Specific gene activation

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Vaijayanthi, T., Sugiyama, H. et al. Adv. Ther. 2023, 6, 2300022

39

Contents
lIntroduction

Contents

lKR12 (DNA alkylating agent with PIP)
lBi-PIP (Brd inhibitor with PIP)
lPIP-HoGu (Integration of PIP and cooperative systems)
lePIP-HoGu (PIP-HoGu with epigenetic modulator)
lSummary & Discussion
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40

Summary
SummaryüPIPs can inhibit binding of transcription factors.
üConjugating PIPs to DNA alkylating agentsenhanced the effectiveness of the alkylating agent.
üConjugating PIPs to epigenetic modulators enabled
genomic loci-selective histone modifications.
üConjugating PIPs to a cooperative binding system
improved DNA sequence selectivity and binding
affinity.
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Discussion
DiscussionüPIPs have the potential to serve as small molecule-based designer
drugs in targeted transcription therapy.
uCell permeability → PIP-HoGu
uConjugation with small molecule drugs (e.g., DNA alkylating
agents)üPIPs are programmable and can be applied to personalized precision
medicine.
uNeed for optimization in each case2024/1/25

Yu, Z., Sugiyama, H. et al. Adv. Drug Delivery Rev. 2019, 147, 66-85

Bai, D., Pandian, G. N. et al. Allergy 2023, 00, 1-18

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Thank you for your kind attention!

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Appendix

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44β-Alanine can improve affinity and relax torsional rigidity
Appendix (KR12)

ü Relaxing torsional rigidity

Ka = 3.7 (±0.9) × 107 M-1β-Alanine
100-foldincreasein affinity

Ka = 3.7 (±1.5) × 109 M-1

l PIP owning over four continuous rings shows high torsional rigidity.
l β-alanine linker = replacement for the Py moiety
Turner, J. M., Dervan, P. B. et al. J. Am. Chem. Soc. 1998, 120(25), 6219–6226

Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215–2235

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Role of indole moiety in KR12
Appendix (KR12)

ü good proximity to the N3 to allow efficient alkylation

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51

KR12 induced p53-dependent cellular senescence
Allele-specific downregulation of
KRAS codon 12 mutant

After KR12 treatmentl a flat cell morphology was displayed in LS180 cellsl SA-b-gal activity was strongly increased→KR12 induced cellular senescence

Appendix (KR12)

Cell cycle arrest
increase of the proportion of G2/M-phase cells

KR12 markedly induced
phosphorylation of p53 at
Ser-15, p21WAF1/CIP1 andphosphorylated histone
variant H2AX (gH2AX). (c) Allele-specific downregulation of KRAS codon 12
mutant: cDNA from the treated RNA was subcloned.
Transfected antibiotic- resistant/colour-selectedcolonies were screened by colony PCR and directcolony-sequencing methods. The number of colonies
for the wild-type or codon 12 KRAS mutant allele ofeach treated group was counted and reported in
Supplementary Table 3. The percentages of wild-type
and mutant KRAS sequences are also shown inparenthesesü KR12 promotes p53-dependent cellular senescence
Hiraoka, K., Sugiyama, H., Nagase, H. et al. Nat. Commun. 2015, 6, 6706

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Effects of linker length of Bi-PIP and position of binding sites
Appendix (Bi-PIP)l Effective position for the acetylation
the sum of acetylation levels of Nuc3–7 << Nuc1
→ synergistic effects of multiple P300s on a single nucleosomel Effects of linker length of Bi-PIP
sufficiently long and flexible linker for the effective
acetylation of H3
Taniguchi, J., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 7108–7115

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Distribution of PIPs in different cell lines
Appendix (Bi-PIP)

l good nuclear uptake→an eight-ring polyamide DNA-binding domain, one or morepositive charges incorporated within either the linker or theturn residue, and a conjugated fluorescein fluorophore
Dervan, P. B. PNAS. 2003, 100, 12063-–12068

Uptake profile of compounds 1–22 in 13 cell lines. + +, Nuclear staining
exceeds that of the medium; +, nuclear staining less than or equal to that of
the medium, but still prominent; -, very little nuclear staining, with the mostfluorescence seen in the cytoplasm and/or medium; --, no nuclear staining.
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Pip-HoGu showed higher binding affinity
Appendix (PIP-HoGu)

EMSA (Gel Shift Assay)
Visualization of band shifts thatform stable complexes-2.32 kcal・mol-1

Figure 3. EMSA illustrating the cooperativity of Pip-HoGu. (A) The
gel-shift behavior of all the positive ODNs with Ada1−Cyd1.
Concentrations are shown in ﬁgure. (B) Quantitative EMSA ofODN2P with Cyd1 at various concentrations (top) and Cyd1
supplemented with saturated Ada1 (bottom). ODNs concentration:
0.1 μM. (C) Equilibrium association constants and free energies forODN2P with Ada1−Cyd1.

Yu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

ü Each bands can be distinguished.
ü Cooperative binding was seen at 0-5 gap= Tm assay
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The cooperative energy of PIP-HoGu was highly distance dependent.
Appendix (PIP-HoGu)

Influence of linker length on cooperative binding
mini-PEG
linkerAda1 = 1 mini-PEG-linker
Ada2 = 2 mini-PEG-linkers

Tm Assayl 0-4 bp gap → Ada2 showed lower stability than Ada1.
(extra-long linker may destabilize the binding affinity)
l 5-6 bp gap → Ada2 showed slightly higher stability
than Ada1. (longer and more flexible linker can makeit easier to form complexes)ü The cooperative energy of PIP-HoGu is highly
dependent on gap distances, and gap >5 bp willdiminish the cooperation even if the linker
length is long enough.

Figure 4. Mechanistic studies of cooperative binding. (A) The gel-shift behavior of all the positive ODNs with Ada2−Cyd1. (B)Tm proﬁles of all positive ODNs in the presence of Ada1−Cyd1 (TmPA, blue, same as Figure 2B) and Ada2−Cyd1 (TmPC, red).

Yu, Z., Sugiyama, H. et al. J. Am. Chem. Soc. 2018, 140, 2426−2429

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Binding dynamics of Cuc1 and Ada3
Appendix (ePIP-HoGu)

Fig. 3 (A and B) Cuc1 binds Ada3 irreversibly in the absence of DNA in an SPR assay. (A) Chemical structureof Ada3–biotin. (B) SPR sensorgram of Cuc1 (125 nM) with multiple rounds of standard injection. One
standard injection consisted of 180 s sample injection, followed by 180 s elution at 20 μL min−1.

Surface plasmon resonance (SPR) assays(B) SPR sensorgram of Cyd1. (C) SPR sensorgram of Cuc1. The sensorgram were normalized to zero at
the start point of injection, even though the interaction is irreversible. Thus, the accurate ka of Cuc1
can not be detected. (D) SPR data in summary. kb of Cuc1 was calculated based on a single injection(125 nM Cuc1) in a new chip. The concentrations were showed in figure. Extensive concentrations of
Cyd1 and Cuc1 were dissolved in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA,
and 0.005 % surfactant P20) with 0.1% DMSO. These solutions were passed over a Ada3-biointylated
chip, in the absence of targeting DNA, immobilized on a sensor chip through a biotin-avidin system.
Kinetic constants were calculated from the surface plasmon resonance sensorgrams for the interactionof guest conjugate Ada-PIP with host Cyd-PIP or CB7-PIP.

Cyd-assisted PIP–HoGu →① the pair binding to DNA, ② the host–guest interactionor the procession of these two steps at a similar rate
CB7-assisted PIP–HoGu →① binding the partner guest, ② synergic DNA binding
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69

Influence of spacing and binding orientation on cooperation
Appendix (ePIP-HoGu)
Fig. 3 (C and D) EMSA illustrating thecooperativity of the CB7-assisted DNA-binding
system. (C) Three binding modes. Positive
binding mode (Mode A) contains series dsDNA(−1P to 8P) with a gap distance (N) ranging from
−1 to 8 bp. Similarly, negative binding mode
(Mode B) includes dsDNA (−1N to 8N) with gap
distance of −1 to 8 bp. (D) The gel-shift
behavior of Modes A, B, and C with Ada3–Cuc1.
ODN concentrations: 1.0 μM. Compoundconcentrations: 10.0 μM. Black arrow: ODNs.
Red arrow: ODNs/Cuc1/Ada3.

Ø EMSA (electrophoretic mobility shift assay)
Cuc1-Ada3
・0-5 bp & 8 bp
・8 bp with partially reversed orientation→ a potent binding affinity

The difference in band-shift with the spacings of 6 bp and 8 bp
l DNA twist angle (host–guest moieties could meet throughcrossing the DNA major groove)l distance between the two PIP-binding sites
l linker length of the two conjugates
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ePIP-HoGu showed high sequence-selectivity
Appendix (ePIP-HoGu)>20 folds selectivity with
2 bp mismatch

2-3 folds selectivity
with 1 bp mismatch

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