Shigeori Takenakatakenaka.che.kyutech.ac.jp/shigeori/Takenaka.pdf · 2017-01-03 · Shigeori...
Transcript of Shigeori Takenakatakenaka.che.kyutech.ac.jp/shigeori/Takenaka.pdf · 2017-01-03 · Shigeori...
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Shigeori TakenakaDepartment of Applied Chemistry,
Research Center for Bio-microsensing Technology,
Kyushu Institute of Technology
International Symposium on Interdisciplinary Education and Research
-Medical Dental and Biotechnological Relationship-
November 11 (Fri), 2016 Kyushu Dental University
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Today’s Topics
2
1. Cancer Diagnosis using
Naphthalene diimde
2. Cancer therapy using
Naphthalene diimide
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Today’s Topics
3
1. Cancer Diagnosis using
Naphthalene diimde
2. Cancer therapy using
Naphthalene diimide
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Voltammogram of ferrocene
Oxi
dat
ion
Curr
ent
Red
uct
ion
Curr
ent
5mA
0 200 400 600
E/mV vs. SCE
1mM Ferrocene
0.1 M LiClO4
in Acetonitrile
- e-+ e-
Fe(II)
Fe(III)
+
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N
NO O
O O
N NHN
N N NH
Fe
Fe
O
O
Electrochemical gene detection using FND
DNA probe immobilized electrode
FND
S. Takenaka et al., Anal. Chem., 72, 1334-1341 (2000).
Hybridization with sample DNA
FNDbinding
Current observed according to the amount of hybridized DNA
Citation 435 from Google Scholar at 20016/11/11
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Cancer markers
intractable cancer
(pancreatic cancer)
lung cancer, stomach cancer Early diagnosis is
established
Early diagnosis is not
established
Cancer diagnoses 982,100 (2015)
number of death 370,900 (2015)
in Japan
decade National Cancer Center in Japan
Cancer Positiverate
diagnostic accuracy
CEA High Low
PSA Low Low
Ras High Low
P53 Low High
Telomerase High High
Telomerase activity may be used as a good marker for early diagnosis of intractable cancer.
mort
alit
y
6
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Telomerase & Telomere DNA
Telomere DNA (Orange parts)
Telomerase : A ribonucleoprotein, synthesizes and directs the telomeric repeats onto the 3’ end. Telomerase activity has been shown to be specifically expressed in cancer cells.
Suggested tetraplex structure
of human telomere
AAUCCCAAUCCCAAUCCC
TTAGGGTTAGGG…
7
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TRAP assay as conventional telomerase activity detection
TRAP (Telomeric Repeat Amplification Protocol) assay
Step 1: Addition of Telomeric Repeats by Telomerase
5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag -3’
5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag ggttag -3’
TS Primer Telomeric Repeats
TS-Telomerase Product
5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag ggttag (ggttag)n -3’
Step 2: Amplification of TS-Telomerase Product by PCR
Step 3: PAGE and Data Analysis (6 bp ladder)
Telomerase activity is evaluated from the number of ladders and their intensity
1 h2.5 h
2 h
Detection(5.5 h)
8
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Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay
Data analysis
Δi: Current increase ratioI : Before reactioni0 : After reaction
Δi (%) = (i-i0)/i0×100i0
i
This assay enabled simple and quick analysis without PCR and gel electrophoresis
We applied this assay to tongue cancer diagnosis
Detection30 min 1 min
S. Sato et al., Anal. Chem., 77, 7304-7309 (2005).Ferrocenylnaphthalene diimide (FND)
9S. Sato, H. Kondo, T. Nojima, S. Takenaka, Analytical Chemistry, 77, 7304-7309 (2005).
N
N
O O
OO
N
N
Fe
Fe
H
H
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Ferrocenylnaphthalene diimide (FND) used in this experiment
N
N
O O
OO
N
NHN
NH
O
O
CH3
CH3
Fe
FeN
N
O O
OO
N
NHN
NH
O
O
CH3
CH3
Fe
Fe N
N
O O
OO
N
NHN
NH
O
O
CH3
CH3
Fe
FeN
N
O O
OO
N
N
CH3
CH3
Fe
Fe
N
N
O O
OO
HN
NH
O
O
Fe
Fe
N N
N N
N
N
O O
OO
HN
NH
O
O
Fe
Fe
N N
N N
N
N
O O
OO
HN
NH
O
O
Fe
Fe
N N
N N
4
N-Methyl-caFc
6
N-Methyl-proFc
3
Piperazine-proFc
5
N-Methyl-acFc
1
Piperazine-caFc
2
Piperazine-acFc
7
N-Methyl-Fc
Different linker chain Different redox potential10
10
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Absorption change of FND 7 upon addition of telomere DNA in the presence of Na+
5 mM FND7, 0.1 M AcONa-AcOH(pH5.5), and 0.1 M NaCl at 25 ℃
0
0.05
0.1
0.15
250 300 350 400 450 500
Ab
so
rba
nce
Wavelength/nm
0 mM telomere DNA
15 mM Telomere DNA
Absorption maximum at 384 nm based on FND7 skeleton showed large hypochromic effect and small red shift with isosbestic point upon addition of telomere DNA.
This suggested the single binding mode of FND 7 with telomere DNA.
11
N
N
O O
OO
N
N
Fe
Fe
H
H
Telomere DNA:
5’-aggg ttaggg ttaggg ttaggg-3’11
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Scatchard Analysis
3
3.5
4
4.5
5
5.5
6
6.5
7
1.2 1.25 1.3 1.35 1.4 1.45
/L
/ 1
05 M
-1
Fitting equation: /L=K(n-)K: binding constant
n: number of the bound FND
molecules per single telomere DNA
Binding constant
Scatchard analysis was carried out by using absorption change at 384 nm in the FND7 solution upon addition of telomere DNA.
1.5 × 106 M-1
Binding number
1.7
The binding constant of FND with telomere DNA was similar to the ligand reported previously.
12
N
N
O O
OO
N
N
Fe
Fe
H
H
12
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Comparison of the binding constant of FND 7 with different DNA structure
0.1 M AcONa-AcOH (pH 5.5) and 0.1 M NaCl at 25 ℃
22-mer oligonucleotide
Binding constant
/10-5M-1
Binding site size
Single stranded DNA5'-CAC CCT ACA CAC CTT CAT CAC C
1.8 2.8
Double stranded DNA5'-CAC CCT ACA CAC CTT CAT CAC C
3'-GTG GGA TGT GTG GAA GTA GTG G
3.1 10.7
Tetraplex DNA5'-AGG GTT AGG GTT AGG GTT AGG G
15.0 1.7
Thermodynamic parameter in the binding of FND with tetraplex DNA
DH=-36.1 kcalmol-1, DS=-92.8 calmol-1K-1
From van’t Hoff plot
10 times
5 times
Entropy control?
13
13
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CD spectra
Small increasing peak at 290 nm and large decreasing peak at 260 nm was obtained upon addition of FND7.
Suggestion of the interaction of FND7 with telomere DNA.
0.1 M AcONa-AcOH (pH 5.5) and 0.1 M NaCl
at 25 oC
In Na+
Typical peaks at 240, 260, and 290 nm based on antiparallel tetraplex DNA structure were identified.
-8
-6
-4
-2
0
2
4
6
250 300 350 400
Elli
pticity /
10
6 d
eg
cm
-1 M
-1
Wavelength/nm
0 mM FND
35 mM FND
14
N
N
O O
OO
N
N
Fe
Fe
H
H
14
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Binding constant of FNDs with telomere DNA in the presence of Na+ or K+ ion
Buffer FND1 FND2 FND3 FND4 FND5 FND6 FND7
Na+ 10-5K 18.4 15.6 27.9 8.6 10.8 112.6 15.0
n 2.6 3.3 2.5 2.5 2.8 1.9 1.7
K+ 10-5K 7.0 6.6 19.2 8.4 18.6 38.6 5.9
n 3.0 4.1 4.2 1.8 1.8 1.4 2.0
In Na+ ionIn K+ ion
N
N
O O
OO
N
NHN
NH
O
O
CH3
CH3
Fe
Fe
6
All FNDs could bind to telomere DNA with high affinity and their binding number were plurality of FNDs bound to single telomere DNA, especially FND6 showed highest affinity.
15
15
Major?
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Telomerase inhibition effect of FND by using TRAP assay
Inhibit conc. FND1 FND2 FND3 FND4 FND5 FND6 FND7
Telomerase/mM 25 25 25 1.0 5.0 5.0 -
PCR/mM - - - 5.0 25 50 -
FND7 does not inhibit the telomerase activity and PCR. FND1-3 only inhibit the telomerase activity. FND4-6 inhibit both reaction.
FND7 was suitable for telomerase assay. FND1-6 might have effective telomerase inhibitor.
16
FND70.5
mM
1.0
mM
5.0
mM
10 m
M
25 m
M
50 m
M
FND1
0.5
mM
1.0
mM
5.0
mM
10 m
M
25 m
M
50 m
M
FND4
0.5
mM
1.0
mM
5.0
mM
10 m
M
25 m
M
50 m
M
16
*TRAP Assay Reaction solution(TRAPeze by Millipore)20 mM Tris-HCl, pH8.3, 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 50 μM each dNTP mixture, 1×TS Primer, 1×Primer Mix, 2 Units Taq polymerase, 250 cells Telomerase, 0.5-50 μM FND 1-7
36 bpInternal Control(PCR ascertainment)
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Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay (ECTA)
Data analysis
Δi: Current increase ratioI : Before reactioni0 : After reaction
Δi (%) = (i-i0)/i0×100i0
i
This assay enabled simple and quick analysis without PCR and gel electrophoresis
We applied this assay to tongue cancer diagnosis
Detection30 min 1 min
S. Sato et al., Anal. Chem., 77, 7304-7309 (2005).
Ferrocenylnaphthalene
diimide (FND)
17K. Mori et al., Clin. Chem., 59, 289 (2013).
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Initial oral cancer
After surgery (resection alone)before surgery
Advancing oral cancer
After surgery (resection + repair operation) QOL decaybefore surgery
hospital charges: ca.
500,000 yen
(surgical cost: ca.
180,000 yen)
length of hospital
stay: ca. 10 days
Five year survival rate
90%
30%
Importance of early diagnosis of oral cancer
hospital charges: ca.
3,000,000 yen
(surgical cost: ca.
2,000,000 yen)
length of hospital
stay: over1.5 months
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It is very
important to
develop early oral
cancer diagnosis.
① Increasing patient under super-aging society
4992
5939
6873
7842
8678
0
2500
5000
7500
10000
1995 2000 2005 2010 2015 2020
Nu
mb
er
of
pat
ien
t
(年)
男 女 計
Future forecast of oral cancer patient
(出所)新図説耳鼻咽喉科・頭頸部外科講座 第5巻 頭頸部腫瘍(2001年、7頁)より
1.7
tim
es
げ
Present oral caner diagnosis
Inefficient
method
② Early diagnosis required medical specialist
Initial Oral Cancer
Hard to discriminate
Stomatitis
(出所) 厚生労働省:統計情報・白書(2010)
③ Limited number of medical specialist (Doctor of dental surgery)
Limited number
Dental surgery
(4.0%)
Dentistry
(87.6%)
Orthodontic
dentistry (3.5%)Child dentistry
(2.0%)
Clinical training
dentistry (2.1%)
Others
(0.7%)
Present state of oral cancer in Japan
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TERT expression in oral cancer cell lines
HSC-2 cells, human oral squamous cell carcinoma cell lines
HSC-3 cells, human tongue squamous cell carcinoma cell lines
Ca9-22 cells, human gingival squamous cell carcinoma cell lines
SAS cells, and human tongue squamous cell carcinoma cells
20
EXPERIMENTAL
1 ) Dissolve samples in 500 μL of lysis buffer, centrifuge.
2) Obtain supernatant of cells.
3) Reverse transcription by Super-Script Reverse TranscriptaseII
(Invitrogen)
4) RT-PCR by FAST SYBR® Green Master Mix
*Internal control: GAPDH gene
oral cancer cell lines
Higher and lower mRNA concentration for the
hTERT, the catalytic subunit gene of telomerase, is
SAS and Ca9-22 cells.
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TRAP assay in oral cancer cell lines
21
Number of
cells SAS Ca9-22 HSC-2 HSC-3
0 - - - -
10 - - - -
50 + - - -
75 + ± ± ±
100 + ± + +
200 + + + +
SAS
ladder=0, -; 0<ladder<5, ±; ladder≧5, +
Detection limit is over 200 cells
TRAP Assay Reaction solution(TRAPeze by Millipore)
20 mM Tris-HCl, pH8.3, 1.5 mM MgCl2, 63 mM KCl, 0.05%
Tween 20, 1 mM EGTA, 50 μM each dNTP mixture, 1×TS
Primer, 1×Primer Mix, 2 Units Taq polymerase, 0, 10, 50, 75, 200 cells
Telomerase reaction, 37 oC, 60 min
40
60
80
100
140
200
I.S.
M
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ECTA in oral cancer cell lines
-S-5’-TTTTTTTTTT AATCCGTCGAGCAGAGTTAGGGTS primer sequenece
Pretreatment of gold electrode: plasma treatment for 30sec
↓
5 nM SS-T8TS1 in 1 mM MCH, 0.1 M NaCl 300 μL, 37 °C, 2 h
↓
i0 measurement (SWV)
0.1 M AcOH-AcOK, 0.1 M KCl (pH 5.5), 20 μM FND
↓
Reaction with cell extract, sample solution 20 μL
[10 ng/μL, 50 mM Tris-HCl (pH 8.0), 1.0 mM MgCl2, 50 mM KCl, 0.10 mM 2-mercaptoethanol,
0.10 M spermidine, 20 μM dNTP mixture]
37 °C, 30 min
↓
i measurement (SWV)
1 min
2 h
1 min
30 min
1 min
Δi (%) = 100×(i – i0)/ i0
SWV: square wave voltammetry 22
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ECTA in oral cancer cell lines
23
SAS ○, Ca9-22 ●HSC-2 ×, HSC-3 □100 Ca9-22 cells
Detection limit is 10 cells (P<0.001).
The detection limit of ECTA was five times lower than that
of TRAP.
0
2
4
6
8
10
0 0.1 0.2 0.3 0.4
Curr
ent/
µA
Potential/V vs. Ag/AgCl
(A) (B)
0
10
20
30
40
50
0 50 100 150 200 250
∆i/
%
Number of cells
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Correlation of the current increase in ECTA with mRNA for correlation
24
The current increase was found to be proportional to the amount of
mRNA for hTERT in the 10-cell lysate quantified by RT-PCR.
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Preparation of clinical samples
Samplesexfoliated buccal cells (B)・・・total oral diagnosis local exfoliated cells (L)・・・local diagnosis
cancer tissue (T)
① Obtain informed consent from all study subjects.
② Collect tissue samples (B, L, T).
③ Dissolve samples in 500 μL of lysis buffer, centrifuge.
④ Obtain supernatant of cells.
⑤ Measure protein concentration in these samples.
⑥ Adjust concentration (TRAP 2000 ng, Electrochemical method 200 ng).
⑦ Detect telomerase activity by TRAP assay or electrochemical assay.
oral squamous cell cancer:24 sampleshealthy individuals:2 samples
Sample preparation
Number of cells:1×107 cells/500 μL
Medical checkup
Cancer suspect patient
25
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Collecting method of exfoliated buccal cells (B)
26
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Collecting method of local exfoliated cells (L)
27
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0
1
2
3
4
5
6
7
0 0.1 0.2 0.3 0.4 0.5
Cu
rren
t/m
A
Potential/V vs. Ag/AgCl
0
1
2
3
4
5
6
7
0 0.1 0.2 0.3 0.4 0.5
Cu
rren
t/m
A
Potential/V vs. Ag/AgCl
SWV results on local exfoliated cells from cancer patient and healthy volunteer
cancer patient healthy volunteer
Cancer patient: current increased after treatmentHealthy volunteer: current unchanged before and after treatment
Telomerase extension with clinical samples was successful on the electrode by FND-based electrochemical assay
Cu
rren
t/μ
A
6
5
4
3
2
1
0
cancer patientheated sample
0 0.1 0.2 0.3 0.4 0.5
Potential/V vs. Ag/AgCl0 0.1 0.2 0.3 0.4 0.5
Potential/V vs. Ag/AgCl
0 0.1 0.2 0.3 0.4 0.5
Potential/V vs. Ag/AgCl
SWV: square wave voltammetry28
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The calculation of the positive rate in ECTA and TRAP
29
0
1
2
3
4
5
6
7
0 0.1 0.2 0.3 0.4 0.5
Cu
rren
t/m
A
Potential/V vs. Ag/AgCl
Classified by Δi
Δi/%<20% -20%≦Δi/%<30% ±Δi/%≧30% +
Classified by number of ladder
ladder =0 -0< ladder <5 ±ladder ≧5, +
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ECTA for clinical samples
30
The change in current
increased more than 30% in
biopsy samples from most
cancer patients, whereas the
increase was 20% or lower in
most healthy individuals.
Cancer Healthy
30%
20%
Telomerase positive
Telomerase negative
BB TT
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0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
TP
F
FPF
Positive “+” : more than19%Negative“-” : less than 19%
Setting up of cut-off value using ROC analysis
Threshold calculation with ROC analysis from ECTA results
Calculation of sensitivity
and specificity
31
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0% 20% 40% 60% 80% 100%
口腔剥離
局所剥離
組織
0% 20% 40% 60% 80% 100%
口腔剥離
局所剥離
組織
TRAP ECTA
Cancer
He
althy
0% 20% 40% 60% 80% 100%
口腔剥離
局所剥離
組織 +
+
+
±
±
±
-
-
- *
+
+
+
±
±
±
-
-
-
0% 20% 40% 60% 80% 100%
口腔剥離
局所剥離
組織
+
+
±
±
±
-
-
-
+
±
±
±
-
-
-
Positive predictive
value
T:50%
L: 45%
B: 14%
Positive predictive
value
T:83%
L: 67%
B: 83%
Negative predictive
value
T:50%
L: 43%
B: 59%
Negative predictive
value
T:83%
L: 68%
B: 81%
ECTA had lower “±” judgment and estimated telomerase activity with more precision.
Calculation of “Positive predictive value using TRAP and ECTA methods
32
T
L
B
T
L
B
T
L
B
T
L
B
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Sensitivity SpecificityPositive
predictivevalue
Negativepredictive
value
T 93% 86% 97% 83%
L 83% 82% 83% 82%
B 93% 86% 97% 83%
Calculation of “Positive predictive value” using ECTA methods
ECTA is not only method carrying high sensitivity and
specificity for tissue, but also for exfoliated buccal cells
(B) and local exfoliated cells (L).33
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Box plot of ECTA result for cancer patient, healthy volunteer, and Leukoplakia, oral lichen planus as benign adenoma
34
0
20
40
60
80
100
120
140
Di/%
Current increase percentage for benign
closed to that for cancer patient rather than
that for healthy volunteer.
Cancer
Benig
n
Healthy
Cancer
Benig
n
Healthy
Cancer
Benig
n
Healthy
Exfoliated buccal cells (B) Local exfoliated cells (L) Cancer tissue (T)
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ConclusionECTA based on FND is more sensitive than
TRAP in terms of detection of cancerous samples taken from tongue cancer patients.
Telomerase activity was detected with exfoliated buccal cells (B) or saliva by ECTA based on FND .
ECTA based on FND will enable early diagnosis of tongue cancer in clinical dentistry and group medical examination.
35
http://takenaka.che.kyutech.ac.jp
K. Mori et al., Clin. Chem., 59, 289 (2013).
M. Hayakawa et al., Electroanalysis, 27, 1159 (2015).
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Today’s Topics
36
1. Cancer Diagnosis using
Naphthalene diimde
2. Cancer therapy using
Naphthalene diimide
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Concept of new type of anti-cancer drug
Telomeric DNA caused G-quadruplex structure to
inhibit the action of telomerase.
Ligand binding to double-stranded DNA causes a
side effect.
Ligands that strongly bind and stabilize G-quadruplex structure are expected to be
highly specific anticancer agents.
Apoptosis
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Fused ring ligand to increase overlap with G-quartet
Porphyrin, perylene diimide, and telomestatin
Increased aromatic ring which can
accommodate with propeller twist of G-quartet
PDC 360A, Phen-DC3, BMVC
Threading intercalator carrying
four substituents
Naphthalene diimide
Telomerase tetraplex DNA binder reported previously
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Development of new telomerase inhibitor-Cyclic intercataor-
Double stranded DNATetraplex DNA
cNDI1NDI-DM
Threading
intercalator
capable to
double stranded
DNA
Control
compound
dsDNA
Cyclic naphthalene diimide Tetraplex DNA specific ligand
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Design and synthesis of cNDI3, 4 as specific teraplex DNA binder
Change to cyclohexane from benzene unit to enhance affinity for tetraplex
DNA and reduce affinity for double stranded DNA.
Introduction of bulky substituents to disturb intercalation for double stranded
DNA.
cNDI3cNDI4
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Interaction analysis of cNDI3, cNDI4 with A-core and dsDNA:UV/Vis spectra measurement under K+
0
0.05
0.1
0.15
0.2
300 350 400 450
Wavelength / nm
Ab
s.
0
0.5
1
1.5
2
2.5
3
0 0.5 1 1.5 2
r/C
r
0
0.05
0.1
0.15
0.2
300 350 400 450
Ab
s.
Wavelength / nm
A-core DNA vs. cNDI3
dsDNA12 vs. cNDI3
A-core DNA vs. cNDI3Scatchard plot
Large
hypochromic shift
was only observed
in cNDI4 upon
addition of A-core!
Binding constant K/ M-1 Selectivity for tetraplex DNA
A-core CT-DNA
NDI-DM 1.4 x 106 3.0 x 105 4.6
cNDI1 3.7 x 106 8.0 x 105 4.6
cNDI2 4.3 x 106 1.4 x 105 31
cNDI3 1.5 x 106 1.3 x 104* 115
cNDI4 6.4 x 106 4.4 x 103* 1600
No hypochromic
shift after addition
of double stranded
DNA
Achievement of
1600-times
selectivity for
tetraplex DNA!
5.0 μM cNDI3
50 mM Tris-HCl (pH 7.4)
100 mM KCl
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42
K=8.6x106 M-1
n=1.9
cNDI4 + a-core cNDI4 + HP27
50 mM potassium or sodium
phosphate buffer (pH7.4).
a-core: 5’-AGGG(TTAGGG)3-3’
HP27: 5’- GCG ATT CTC GGC TTT
GCC GAG AAT CGC-3’
Isothermal Titration Calorimetry (ITC) measurements of cNDI4 with tetraplex DNA
and double stranded DNA
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TRAP activity was determined with 500 ng of an extract of a TRAPeze positive control cell line. IC50s were determined as
follows: ligand concentration under half telomerase activity with no ligand.
Telomerase inhibition by cNDIs in a TRAP assaycNDI3 NDI-DMcNDI4
IC50=0.5 μM IC50= 3.0μM IC50=3.0 μM
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Computer modeling of the complex of cNDI4 or cNDI3 with A-core.
cNDI4 cNDI3
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46
IC50/mM
Doxorubicin 1.48
cNDI-3 0.17
cnDI-4 0.28
WST-1 Cell Proliferation Assay (EMT6)
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ConclusionCyclic naphthalene diimide, cNDI4 was newly
synthesized aiming at effective anticancer drug.
cNDI4 have the binding affinity of 106 M-1 for tetraplex DNA.
cNDI4 shows 1600-times higher affinity for tetraplex DNA than for double stranded DNA.
cNDI4 inhibits telomerase activity with stabilized the tetraplex DNA of telomere DNA.
47
http://takenaka.che.kyutech.ac.jp
Yugo Esaki et al., Chem. Commun., 50: 5967 (2014).
Md. Monirul Islam et al., Bioorg. & Med.Chem., 23, 4769 (2015).
Md. Monirul Islam et al., Molecules, 20(6), 10963 (2015).
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48
Telome-Chyan
Thank for your kind attention !
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49
DNA10-5K/M-1 (n)
cNDI1 cNDI2 NDI-DM
a-core (K+) 15 (1.6) 86 (1.9) 16 (1.5)
a-core (Na+) 1.1* 2.2* 1.1*
dsOligo (K+) 0.30* 0.33* 6.0 (2.8)
dsOligo (Na+) - - 6.0 (3.0)
50-
times
2.7-
times
-Binding parameter and effect for Tm-
DNA Tm/˚CΔTm/˚C
1 2 3
a-core (K+) 68 2.0 3.0 6.0
a-core (Na+) 56 1.0 1.3 1.0
dsOligo (K+) 58 0 0 5.0
dsOligo (Na+) 61 0 0 5.0
cNDI1 has 260-times higher affinity for tetraplex DNA than for double stranded one.
Structural stabilization effect of cNDI1, cNDI2 is not observed for double stranded DNA.
Binding affinity of cNDI1 & 2 with a-core or dsDNA12