Sequence-specific base pair mimics are efficient topoisomerase IB inhibitors

Article abstract of DOI:10.1021/bi2012959

Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3'-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA- topoisomerase-inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I-DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequencespecific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity.

Full text of DOI:10.1021/bi2012959

Article
pubs.acs.org/biochemistry
Sequence-Specific Base Pair Mimics Are Efficient Topoisomerase IB
Inhibitors
Pierre Vekhoff,†,‡ Maria Duca,§ Dominique Guianvarc’h,∥ Rachid Benhida,§ and Paola B. Arimondo*,†,‡
†CNRS UMR7196, Museu
́
m National d’Histoire Naturelle, 43 rue Cuvier, 75005 Paris, France
‡INSERM U565, 43 rue Cuvier, 75005 Paris, France
§CNRS UMR6001 LCMBA, Institut de Chimie de Nice, Universite
France
́
de Nice-Sophia Antipolis, Parc Valrose, 06108 NICE Cedex 2,
∥UPMC Paris 06-ENS-CNRS, UMR 7203, Laboratoire des Biomolec
́
ules and FR2769 Chimie Moleculaire, Universite Pierre et Marie
́
́
Curie, 4 place Jussieu, 75005 Paris, France
S
* Supporting Information
ABSTRACT: Topoisomerase IB controls DNA topology by
cleaving DNA transiently. This property is used by inhibitors,
such as camptothecin, that stabilize, by inhibiting the religation step,
the cleavage complex, in which the enzyme is covalently attached to
the 3′-phosphate of the cleaved DNA strand. These drugs are used
in clinics as antitumor agents. Because three-dimensional structural
studies have shown that camptothecin derivatives act as base pair
mimics and intercalate between two base pairs in the ternary DNA−
topoisomerase−inhibitor complex, we hypothesized that base pairs
mimics could act like campthotecin and inhibit the religation
reaction after the formation of the topoisomerase I−DNA cleavage
complex. We show here that three base pair mimics, nucleobases
analogues of the aminophenyl-thiazole family, once targeted specifically
to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-
specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage
complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was
observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex
formation is the essential feature for the inhibition activity.
DNA topoisomerases control DNA topology through a transient
cleavage of the phosphate backbone.1 Type I topoisomerases
(topo I) act by cleaving only one strand of the DNA, while type
II topoismerases (topo II) act by cleaving both DNA strands. In
all cases, the enzyme forms a transitory cleavage complex in
which the catalytic tyrosine is covalently linked to the phosphate
of the DNA at the cleavage site, leaving a free OH end. Topo-
isomerases are ubiquitous and essential for cells.2 In human
cells, topo IB and topo II are the target of commonly used anti-
tumor agents such as camptothecin (CPT)3 and etoposide,4
respectively. These agents act by stabilizing the cleavage complex,
mostly by inhibiting the religation reaction that maintains the
integrity of the DNA genome. Thus, they generate DNA strand
breaks that poison the cell and induce cell death. In 2002, the
crystal structure of the ternary complex of human topo IB co-
valently linked to DNA in the presence of topotecan, a deri-
vative of CPT, was determined5 (Protein Data Bank entries
1K4S and 1K4T). The drug was found intercalated in the DNA
and behaved as a base pair increasing the distance between the
base pair at position −1 of the cleavage site and the one at posi-
tion +1 from 3.6 to 7.2 Å with a twist of 10°. This increased the
distance between the 5′-OH group of the downstream cleaved
DNA strand from the 3′-tyrosylphosphoryl bond that keeps the
DNA attached to the enzyme, thus inhibiting the religation
reaction. Interestingly, according to the authors, the intercalation
can occur only after the cleavage complex between the topo IB
and the DNA is formed. Several other inhibitors, even of different
chemical families, have been crystallized in the cleavage complex,
and all are found intercalated in the DNA at the cleavage site.6,7
In the past, we developed sequence-specific topoisomerase
poisons upon covalent attachment of the inhibitors to sequence-
specific DNA ligands, such as triplex-forming oligonucleotides
(TFOs) or minor groove ligands.8,9 We demonstrated that the
DNA ligand positions the inhibitors specifically in the proximity
of its binding site and stabilizes its interaction with the DNA−
topoisomerase cleavage complex, leading to sequence-specific
DNA cleavage.10−13 It is worth noting that we applied this
strategy to inhibit site-specifically topo IB and induce a sequence-
specific DNA cleavage using an inactive camptothecin derivative
Received: August 16, 2011
Revised: November 8, 2011
Published: November 29, 2011
© 2011 American Chemical Society
43
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
Figure 1. (A) Model proposed for the specific recognition of an AT base pair by nucleobase S. (B) Nucleobase analogues synthesized in this work.
(C) Chemical structures of the TFO conjugates, where P stands for 5-propynyldeoxyuridine and M for 5-methyldeoxycytidine. (D) Sequences of the
TFO and of the DNA target (the triplex site is underlined). The topo IB cleavage site is colored red.
(i.e., lacking one potential H-bond in the cleavage complex).14
This showed that it is possible to render a molecule otherwise
inactive into a good and specific topo IB inhibitor by conjuga-
tion to a DNA ligand, such as a TFO.
because it also recognizes CG, depending on the pH and the
triplex sequence, which maintain the H-bonding network and
the cooperativity of the Hoogsteen base pairing. This low
selectivity was attributed to an intercalative mode of recognition.
Therefore, we expected that S intercalation would be highly
favored when S is attached to an oligonucleotide via an ended
linker by abolishing the H-bonding network.
Because modified nucleobases of S could act as base pair
mimics, we hypothesized that they could fit perfectly in the
DNA−topo IB cleavage complex, when positioned by a DNA
ligand, and fulfill the requirements for an intercalating poison
as observed for the camptothecin analogues in the crystal
structure.5,6 We thus designed and synthesized triplex-forming
oligonucleotides conjugated to artificial nucleobases 1, 4, and 7
(Figure 1B) inspired by the structure of S nucleoside, via linker
with different lengths [(CH2)n where n = 4 or 6 (conjugates
TFO-10−TFO-15 in Figure 1C)]. We then studied their
binding properties with respect to the target duplex as well as
The formation of a DNA triplex with oligonucleotides is
limited to oligopurine·oligopyrimidine sequences, and in an effort
to overcome this limitation and to improve the stability toward
mixed purine/pyrimidine target sequences, a number of artificial
nucleic acids have been designed and synthesized.15−21 In this
context, we have demonstrated that a novel nucleoside
analogue [S (Figure 1A)], when incorporated into a pyrimidine
motif TFO, can effectively circumvent a purine·pyrimidine base
pair interruption in an oligopyrimidine·oligopurine sequence.16,22
Indeed, S is an extended heterocyclic ring system (substituted
aminophenylthiazole) previously designed for the recognition
of inverted AT base pairs by specific Hoogsteen H-bonding in
the major groove of the DNA duplex (Figure 1A). Several
structure−affinity assays argue for this mode of interaction.16,22
However, in some cases, nucleobase S exhibits less selectivity
44
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
their ability to inhibit topo IB activity in a sequence-specific
manner.
under reduced pressure followed by flash chromatography (97:3
CH2Cl2/acetone) led to 1.41 g of compound 6 (92% yield) ob-
1
tained as a yellow solid: Rf = (1:1 CH2Cl2/methanol); H NMR
EXPERIMENTAL PROCEDURES
(DMSO-d6) δ 2.41 (s, 3H), 7.38 (d, 2H, J = 8.2), 7.70−7.90 (m,
1H), 8.02 (s, 1H), 8.07 (d, 2H, J = 8.2), 8.15−8.22 (m, 1H), 8.35−
8.45 (m, 1H), 8.59 (m, 1H), 8.83 (s, 1H); 13C NMR (DMSO-d6) δ
21.5, 111.4, 120.6, 122.7, 124.3, 128.6, 129.4, 129.5, 130.7, 132.2,
136.3, 143.4, 147.0, 148.7, 150.0, 159.4, 165.6; mass spectrum (ESI)
m/z 340.07471 (M + H)+ (C17H14N3O3S requires m/z 340.07559).
2-N-Benzoylamino-4-(3-aminophenyl)thiazole (7). A
solution of compound 6 (1.41 g, 4.15 mmol) in a mixture of
acetic acid and ethanol (1:1, v/v, 50 mL) was stirred under a
hydrogen atmosphere in the presence of palladium on activated
carbon (10%). The mixture was stirred for 3 h. After removal of
the catalyst by filtration through a pad of Celite, the filtrate was
concentrated under reduced pressure and the product crystal-
lized in ethyl ether to give 1.22 g of pure compound 7 (95%
yield) obtained as a white solid: Rf = 0.54 (9:1 CH2Cl2/
■
Chemistry. All oligonucleotides were purchased from
Eurogentec (Seraing, Belgium). The duplex target dsDNA
has the following sequence (the triplex site is underlined):
5′-CACTCCCTATCAGTGATAGAGAGAGAGAAAAAAAGA-
GAAGATCTGAGCTCGGTACCCT-3′/3′-GTGAGGGATA-
GTCACTATCTCTCTCTCTTTTTTTCTCTTCTAGACT-
CGAGCCATGGGA-5′. Solvents and most of the starting
materials were purchased from Aldrich and Alfa Æsar. High-
resolution mass spectra (HRMS) were obtained with a LTQ
Orbitrap hybrid mass spectrometer with an electrospray
ionization probe (Thermo Scientific, San Jose, CA) by direct
infusion from a pump syringe to confirm the correct molar
mass and high purity of compounds. The H and 13C NMR
1
(200 and 50 MHz, respectively) spectra were recorded on a
Bruker AC 200 spectrometer. Chemical shifts are expressed as
parts per million from tetramethylsilane. Splitting patterns have
been designated as follows: s (singlet), d (doublet), t (triplet),
and m (multiplet). Coupling constants (J values) are listed in
hertz. Reactions were monitored by analytical thin-layer
chromatography, and products were visualized by exposure to
UV light. VWR silica gel (230−400 mesh) was used for column
chromatography. Dichloromethane, methanol, cyclohexane, and
ethyl acetate employed as eluents in column chromatography
were distilled on a rotary evaporator prior to being used. High-
performance liquid chromatography analysis was conducted on a
reverse phase column (Thermo, RP C18, 250 mm × 4.6 mm,
5 μm) using a 0 to 100% gradient of water and acetonitrile
with UV detection at 260 nm.
1
MeOH); H NMR (DMSO-d6) δ 2.20 (s, 3H), 6.60−6.68
(m, 1H), 7.15−7.25 (m, 3H), 7.46 (d, 2H, J = 8.2), 7.56 (s, 1H),
8.15 (d, 2H, J = 8.2); 13C NMR (DMSO-d6) δ 21.5, 108.0, 111.7,
114.0, 114.1, 128.6, 129.5, 129.7, 135.4, 143.1, 149.2, 150.3, 158.7,
165.5; mass spectrum (ESI) m/z 310.10144 (M + H)+
(C17H16N3OS requires m/z 310.10141).
General Procedure for the Synthesis of 5-Bromovaleric
Acid (8) and 7-Bromoheptanoic Acid (9). To a solution of
ethyl 5-bromovalerate or ethyl 7-bromoheptanoate (1.30 mmol)
in methanol (5 mL) was added 2 N aqueous LiOH (0.6 mL,
1.30 mmol), and the mixture was stirred at room temperature
for 30 min. The reaction mixture was then concentrated, redis-
solved in ethyl acetate, washed with 2 N aqueous HCl and brine,
and finally dried over MgSO4. Filtration and concentration under
vacuum gave the crude products 8 and 9, respectively, as white
solids. These compounds were then used directly in the follow-
ing reactions without further purification.
General Procedure for the Synthesis of Compounds 10,
12, and 14. To a solution of 5-bromovaleric acid (8, 150 mg,
0.742 mmol) in anhydrous dichloromethane (5 mL) were
added HOBt (100 mg, 0.742 mmol) and EDAC (142 mg,
0.742 mmol). After the mixture had been stirred at room
temperature under argon for 20 min, compound 4, 7, or 10 was
added (0.485 mmol), and the mixture was stirred for an addi-
tional 3 h. The reaction mixture was then washed twice with
water (2 × 15 mL) and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography (1:1
cylclohexane/EtOAc) led to compounds 10, 12, and 14 in 65,
70, and 68% yields, respectively.
2-N-Acetylamino-4-(4-nitrophenyl)thiazole (3). To a
solution of 1-bromo-4-nitroacetophenone (2, 2.5 g, 10 mmol)
in ethanol (60 mL) was added the N-acetylthiourea (1 equiv,
1.24 g). The reaction mixture was stirred at 80 °C for 30 min
and then left to cool to room temperature. The precipitate was
filtered and washed with an ethanol/ether mixture leading to
2.2 g of compound 3 (98% yield) obtained as a yellow solid:
1
Rf = 0.7 (9:1 CH2Cl2/MeOH); H NMR (DMSO-d6) δ 2.17
(s, 3H), 7.70 (s, 1H), 8.13 (d, 2H, J = 9.0), 8.30 (d, 2H, J =
9.0); 13C NMR (DMSO-d6) δ 22.9, 112.7, 124.6, 126.8, 140.7,
146.8, 146.9, 158.9, 169.2; mass spectrum (ESI) m/z 264.04376
(M + H)+ (C11H10N3O3S requires m/z 264.04429).
2-N-Acetylamino-4-(4-aminophenyl)thiazole (4). A solu-
tion of compound 3 (2.20 g, 8.37 mmol) in a mixture of acetic
acid and ethanol (1:1, v/v, 50 mL) was stirred under a hydro-
gen atmosphere in the presence of palladium on activated carbon
(10%) for 3 h. After removal of the catalyst by filtration through a
pad of Celite, the filtrate was concentrated under reduced pressure
and the product crystallized in ethyl ether to give 1.85 g of pure
compound 4 (95% yield) obtained as a white solid: Rf = 0.54
N-[3-(2-Acetamidothiazol-4-yl)phenyl]-5-bromopentanamide
(10): Rf = 0.25 (1:1 cyclohexane/EtOAc); 1H NMR (DMSO-d6)
δ 1.65−1.90 (m, 4H), 2.16 (s, 3H), 2.36 (t, 2H, J = 7.0), 3.57
(t, 2H, J = 6.4), 7.30−7.40 (m, 2H), 7.50−7.60 (m, 2H), 8.28
(s, 1H); 13C NMR (DMSO-d6) δ 22.9, 24.1, 32.1, 35.2, 35.6,
108.3, 117.1, 119.0, 121.0, 129.3, 135.1, 140.0, 149.1, 158.3,
169.0, 171.3; mass spectrum (ESI) m/z 398.03522 (M + H)+
(C15H19N3O281BrS requires m/z 398.03554).
1
(9:1 CH2Cl2/MeOH); H NMR (DMSO-d6) δ 2.15 (s, 3H),
5.26−5.28 (br s, 2H), 6.60 (d, 2H, J = 8.6), 7.17 (s, 1H), 7.56
(d, 2H, J = 8.4); 13C NMR (DMSO-d6) δ 22.9, 103.5, 114.1,
122.8, 127.0, 148.9, 150.1, 157.8, 168.7; mass spectrum (ESI) m/z
234.06985 (M + H)+ (C11H12N3OS requires m/z 234.07011).
2-N-Benzoylamino-4-(3-nitrophenyl)thiazole (6). To a
solution of 2-amino-4-(4-aminophenyl)thiazole (5, 1.0 g,
4.52 mmol) in anhydrous pyridine (40 mL) was added 4-
methylbenzoyl chloride (1.2 equiv, 720 μL, 5.43 mmol). The
mixture was stirred for 4 h at room temperature, washed twice
with water (2 × 20 mL), and dried over MgSO4. Concentration
N-[4-(2-Acetamidothiazol-4-yl)phenyl]-5-bromopentan-
1
amide (12): Rf = 0.46 (1:1 cyclohexane/EtOAc); H NMR
(DMSO-d6) δ 1.65−1.90 (m, 4H), 2.15 (s, 3H), 2.36 (t, 2H, J =
7.0), 3.57 (t, 2H, J = 6.4), 7.47 (s, 1H), 7.64 (d, 2H, J = 7.6),
7.81 (d, 2H, J = 8.8); 13C NMR (DMSO-d6) δ 22.9, 24.1, 32.1,
35.2, 35.7, 106.3, 119.5, 126.4, 129.6, 139.2, 148.9, 158.2, 169.0,
171.2; mass spectrum (ESI) m/z 398.03519 (C16H19O2N381BrS
requires m/z 398.03554).
45
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
a
Scheme 1.
a
Reagents: (a) N-acetylthiourea, EtOH, 80 °C, 30 min; (b) H2, Pd/C, AcOH/EtOH (1:1, v/v), 3 h; (c) 4-methylbenzoyl chloride, pyridine, 4 h.
N-{4-[3-(5-Bromopentanamido)phenyl]thiazol-2-yl}-4-
methylbenzamide (14): Rf = 0.59 (1:1 cyclohexane/EtOAc);
1H NMR (DMSO-d6) δ 1.65−1.90 (m, 4H), 2.30−2.40 (m,
5H), 3.56 (t, 2H, J = 6.4), 7.47 (s, 1H), 7.30−7.45 (m, 4H), 7.55−
7.65 (m, 2H), 8.04 (d, 2H, J = 8.2), 8.33 (s, 1H); 13C NMR
(DMSO-d6) δ 21.1, 23.5, 24.1, 32.1, 33.0, 35.1, 35.6, 109.0, 117.2,
119.1, 121.1, 128.6, 129.2, 129.5, 135.3, 140.0, 143.2, 149.5, 159.0,
165.5, 171.2, 174.6; mass spectrum (ESI) m/z 474.06671
(C22H23O2N381BrS requires m/z 474.06684).
Synthesis of the Conjugates. The conjugates were syn-
thesized from the 3′-thiophosphate TFO as described pre-
viously.23 TFO-NCpt is described in ref 23.
All conjugates were analyzed by UV spectrophotometry and
mass spectrometry. TFO-10: ES-MS [M − H]− found 5475.0,
calcd 5474.1. TFO-11: ES-MS [M − H]− found 5503.9, calcd
5502.7. TFO-12: ES-MS [M − H]− found 5475.8, calcd 5474.1.
TFO-13: ES-MS [M − H]− found 5502.7, calcd 5502.7. TFO-
14: ES-MS [M − H]− found 5551.1, calcd 5550.2. TFO-15: ES-
MS [M − H]− found 5579.2, calcd 5578.2.
Biology. Topo IB-Mediated DNA Cleavage. Sequence-
specific topo 1B-mediated DNA cleavage was assessed as described
in ref 11 at 30 °C and pH 7.2.
General Procedure for the Synthesis of Compounds
11, 13, and 15. To a solution of 7-bromoheptanoic acid
(9, 200 mg, 0.965 mmol) in anhydrous dichloromethane (5 mL)
were added HOBt (130 mg, 0.965 mmol) and EDAC (185 mg,
0.965 mmol). After the mixture had been stirred at room tempera-
ture under argon for 20 min, compound 4, 7, or 10 was added
(0.644 mmol), and the mixture was stirred for an additional 3 h.
The reaction mixture was then washed twice with water (2 × 15 mL)
and dried over MgSO4. Concentration under reduced pressure
followed by flash chromatography (1:1 cyclohexane/EtOAc1) led to
compounds 11, 13, and 15 in 75, 73, and 80% yield, respectively.
N-[3-(2-Acetamodothiazol-4-yl)phenyl]-7-bromoheptan-
DNase I Footprinting. The samples were prepared for topo
IB-mediated DNA cleavage and DNase I digestion, and analysis
was conducted as described in ref 24.
Fluorescence Intercalation Displacement Assay (FID). Each
well of a 96-well plate was loaded with Tris buffer containing ethi-
dium bromide {130 μL of a 0.575 μM solution in buffer [0.05 M
Tris, 100 mM NaCl, and 10 mM MgCl2 (pH 7.4)]}. To each well
was added 10 μL of previously hybridized dsDNA (final
concentration of 0.500 μM) followed by the compound (10 μL
of a solution in Tris buffer at increasing concentrations of 1.5, 7.5,
112.5, 300, and 1500 μM, leading to final concentrations of 0.1,
0.5, 7.5, 20, and 100 μM, respectively). When TFO was added, the
protocol was slightly modified. Tris buffer was added first (110 μL
of 0.05 M Tris, 100 mM NaCl, and 10 mM MgCl2) followed by
DNA (10 μL, final concentration of 0.5 μM) and TFO (10 μL,
final concentration of 5 μM). After incubation for 2 h at 25 °C to
yield the triplex, ethidium bromide was added (10 μL, 7.5 μM
solution in Tris buffer, final concentration of 0.5 μM) followed by
the compound to be analyzed (10 μL of a solution in Tris buffer at
increasing concentrations of 1.5, 7.5, 112.5, 300, and 1500 μM,
leading to final concentrations of 0.1, 0.5, 7.5, 20, and 100 μM,
respectively). In both cases, after incubation at 25 °C for 30 min,
each well was read (average of 20 readings) on a GeniosPro Tecan
fluorescence plate reader (excitation at 515 nm, emission at 595 nm)
in duplicate with two control wells (no agent, 100% fluorescence; no
DNA, 0% fluorescence). Fluorescence readings are reported as the
percentage of fluorescence relative to control wells.
1
amide (11): Rf = 0.33 (1:1 cyclohexane/EtOAc); H NMR
(DMSO-d6) δ 1.65−1.90 (m, 4H), 2.16 (s, 3H), 2.36 (t, 2H, J =
7.0), 3.57 (t, 2H, J = 6.4), 7.30−7.40 (m, 2H), 7.50−7.60
(m, 2H), 8.28 (s, 1H); 13C NMR (DMSO-d6) δ 22.9, 24.1, 32.1,
35.2, 35.6, 108.3, 117.1, 119.0, 121.0, 129.3, 135.1, 140.0, 149.1,
158.3, 169.0, 171.3; mass spectrum (ESI) m/z 424.06891
(C18H23O2N3BrS requires m/z 424.06889).
N-[4-(2-Acetamodothiazol-4-yl)phenyl]-7-bromoheptan-
1
amide (13): Rf = 0.45 (1:1 cyclohexane/EtOAc); H NMR
(DMSO-d6) δ 1.65−1.90 (m, 4H), 2.16 (s, 3H), 2.36 (t, 2H, J =
7.0), 3.57 (t, 2H, J = 6.4), 7.30−7.40 (m, 2H), 7.50−7.60 (m,
2H), 8.28 (s, 1H); 13C NMR (DMSO-d6) δ 22.9, 24.1, 32.1,
35.2, 35.6, 108.3, 117.1, 119.0, 121.0, 129.3, 135.1, 140.0, 149.1,
158.3, 169.0, 171.3; mass spectrum (ESI) m/z 426.06662
(C18H23O2N381BrS requires m/z 426.06684).
N-{4-[3-(7-Bromoheptanamido)phenyl]thiazol-2-yl}-4-
methylbenzamide (15): Rf = 0.61 (1:1 cyclohexane/EtOAc);
1H NMR (DMSO-d6) δ 1.30−1.45 (m, 4H), 1.60−1.70 (m, 2H),
1.75−1.85 (m, 2H), 2.33 (t, 2H, J = 7.3), 2.40 (s, 3H), 3.54 (t, 2H,
J = 6.7), 7.30−7.45 (m, 4H), 7.55−7.65 (m, 2H), 8.04 (d, 2H, J =
8.2), 8.32 (s, 1H); 13C NMR (DMSO-d6) δ 16.4, 21.4, 25.3, 27.7,
28.1, 32.5, 35.6, 36.6, 109.0, 117.2, 119.0, 121.0, 128.6, 129.5,
135.2, 140.0, 143.2, 149.6, 150.9, 152.9, 158.9, 165.5, 171.6, 174.6;
mass spectrum (ESI) m/z 502.09811 (C24H27O2N381BrS requires
m/z 502.09814).
RESULTS
■
Synthesis of Nucleobase Derivatives 1, 4, and 7
(Scheme 1). The three nucleobases chosen for this study are
reported in Figure 1B. Compound 1 [2-acetamido-4-(3-
aminophenyl)thiazole] corresponds to the previously published
46
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
a
Scheme 2.
a
Reagents: (a) LiOH, methanol/water, 30 min; (b) HOBt, EDAC, CH2Cl2, 3 h.
nucleobase of nucleoside S and has been synthesized following
Cleavage and Binding Activity. The conjugates and
nucleobases were tested for their ability to inhibit topo IB. As
shown in Figure 2A, the conjugates, once bound to the DNA
[DNase I footprinting (Figure 2A, lanes 18−25)], induced an
efficient and sequence-specific DNA cleavage mediated by topo
IB on the 3′ side of the triplex at a T↓T site (arrow, lanes 7, 9,
11, 13, 15, and 17) like the corresponding conjugate of a cam-
ptothecin derivative, TFO-NCpt (lane 5), used as a control.
DNA cleavage was observed at concentrations as low as 0.05 μM
(as shown for TFO-10 in Figure 2B, lane 8). The conjugates
showed a comparable efficacy (between 10 and 30%, depending
on the DNA fragment preparation). The length of the linker
had a small effect on the cleavage efficiency of conjugates TFO-
15 and TFO-14 with a mean increase of ∼20% in the cleavage
for the six-carbon linker (TFO-15). The nature of the nucleo-
base had little effect; still the best was nucleobase 7, bearing an
aromatic substituent, present in conjugates TFO-15 and TFO-
14. The nucleobase analogues alone 20 times more con-
centrated than the conjugates (Figure 2A, lanes 6, 8, 10, 12, 14,
and 16) are weak inhibitors, while the TFO alone induced little
modification (lane 3).
the reported procedure, starting from commercial 3-nitro-
acetophenone.16,22,25 Compounds 4 and 7 are new analogues of
1, conceived to mimic a base pair and useful for studying the
influence of the position and nature of the substituents of the
nucleobase on the interaction with target DNA. Compound 4
[2-acetamido-4-(4-aminophenyl)thiazole] is an analogue of 1
but bears the amino group in the para instead of in the meta
position. This derivative has been synthesized following the
procedure used for compound 1 (Scheme 1A). In this case, the
1-bromo-4-nitroacetophenone (2) is coupled with N-acetylth-
iourea in ethanol leading to the 2-N-acetylamino-4-(4-
nitrophenyl)thiazole (3) in 98% yield. The following reduction
of the nitro group by catalytic hydrogenation led to the desired
compound 4 in 95% yield. The third nucleobase [7 (Scheme 1B)]
was obtained in two steps starting from known 2-amino-4-
(3-nitrophenyl)thiazole (5) by reaction of the amino group in
the presence of benzoyl chloride in dichloromethane to yield 2-
N-benzoylamino-4-(3-nitrophenyl)thiazole (6) in 92% yield.
Compound 7 was obtained in 95% yield by catalytic hydro-
genation in the presence of palladium on carbon.
The synthesized nucleobases 1, 4, and 7 were conjugated to a
TFO to study their ability to inhibit topo IB when positioned
site-specifically on the DNA sequence. The DNA target se-
quence, its corresponding TFO, and the geometry and structure
of the cleavage site have been optimized previously.26 The
distance between the nucleobase and the oligonucleotide chain
was modulated using two appropriate linkers of four and six
atoms (Scheme 2) according to previous results.11 5-Bromovaleric
acid (8) and 7-bromoheptanoic acid (9), obtained after hydrolysis
of commercially available ethyl esters in the presence of LiOH in
methanol, were coupled with the nucleobases in the presence of
HOBt and EDAC in dichloromethane. This led to the desired
amide compounds 10−15 in 65−80% yield, ready to be coupled
to the TFO.
Synthesis of TFO−Nucleobase Conjugates. The conju-
gates between compounds 10−15 and the 16-mer TFO illu-
strated in Figure 1C (TFO-10−TFO-15) were synthesized
following the procedure that we have recently optimized.23 To
increase the stability of the triple-helical structure at pH 7.2 and
37 °C, thymidine was substituted with 5-propynyldeoxyuridine
(P) and deoxycytidine with 5-methyldeoxycytidine (M).10,27
The DNA target sequence is reported in Figure 1C: the TFO
binding site is underlined and the topo I cleavage site colored
red.
DNase I footprinting confirmed that the conjugates are
bound to the DNA target (Figure 2A, lanes 20−25). In addition,
there is a clear increase in the digestion pattern of the DNase I at
the 3′ end of the triplex−duplex junction where the nucleobases
are positioned by the TFO. This exaltation is not observed in the
presence of the triplex alone (lane 19). This seems to suggest
that the base mimics are intercalated at the 3′ triplex−duplex
junction28 and thus could exercise their poisoning activity of topo
IB in the same manner that was demonstrated for campthotecin
analogues. To test the binding of the base mimics to DNA, we
performed fluorescence intercalation displacement assays
(Figure 3). Figure 3A shows the binding of compound 14 to
the DNA occurs only once the triplex was formed on the DNA
target. The same was valid for all compounds (Figure 3B), while
the compounds alone bound only weakly to the DNA target as
assessed by DNase I footprinting experiments (Figure 3C).
Finally, we investigated the effect of the conjugation of the
TFO to 10−15 on the stability of the triplex. C50 values were
determined by measuring DNA binding of the TFO alone or
conjugated to the radiolabeled DNA target in gel shift analysis.
While camptothecin conjugates destabilize the triple-helical
structure,8 these compounds only slightly alter the stability of
the triplex as shown by the C50 values listed in Table 1.
47
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
Figure 2. DNA cleavage assays with topo IB and DNase I footprinting. Cleavage products were resolved on a 8% polyacrylamide gel containing 7 M
urea. Adenine/guanine-specific Maxam−Gilbert chemical cleavage reactions were used as markers (lane G+A). (A) The target duplex alone (lane 1)
was incubated with 10 units of topo IB (lane 2) and in the presence of 5 μM TFO (lane 3), 10 μM Cpt (lane 4), 5 μM TFO-NCpt (lane 5), 100 μM
14 (lane 6), 5 μM TFO-14 (lane 7), 100 μM 15 (lane 8), 5 μM TFO-15 (lane 9), 100 μM 12 (lane 10), 5 μM TFO-12 (lane 11), 100 μM 13 (lane 12),
5 μM TFO-13 (lane 13), 100 μM 10 (lane 14), 5 μM TFO-10 (lane 15), 100 μM 11 (lane 16), or 5 μM TFO-11 (lane 17). The DNA target was
digested with DNase I (lane 18) after incubation with 5 μM TFO (lane 19), TFO-14 (lane 20), TFO-15 (lane 21), TFO-12 (lane 22), TFO-13
(lane 23), TFO-10 (lane 24), or TFO-11 (lane 25). (B) The target duplex alone (lane 1) was incubated with 10 units of topo IB (lane 2) and in the
presence of 10 μM Cpt (lane 3), 5 μM TFO-NCpt (lane 4), 1, 0.5, 0.1, and 0.05 μM TFO-10 (lanes 5−8, respectively), or 1 and 100 μM 10 (lanes 10
and 9, respectively).
formation.16 This derivative is able not only to form three specific
hydrogen bonds with the TA base pair but also to mimic a base
The determination of the crystal structure of the DNA−topo
pair when integrated into a triple-helical structure. On the other
DISCUSSION
■
IB−topotecan ternary complex5 prompted us to design new
hand, we have demonstrated that 20-deoxycamptothecin, lacking
topoisomerase inhibitors that bear the same features observed
an important interaction for the stabilization of the topo IB
for camptothecin derivatives: the molecule should (i)
cleavage complex and thus resulting in an inactive inhibitor of topo
intercalate as a base pair in the target DNA and (ii) be kept
firmly in the cleavage complex. On one hand, nucleoside S
(Figure 1A), bearing an aminophenyl-thiazole derivative as a
nucleobase, was developed by our group to recognize a TA inver-
sion in a DNA oligopurine·oligopyrimidine sequence during triplex
IB, could become again a topo IB poison upon conjugation to a
TFO.14 This is due to the fact that the DNA ligand moiety of the
conjugates binds to the DNA at its target site,29,30 positions the
drug at this site, and keeps it in the cleavage complex, thereby
inducing a specific topo IB-mediated DNA cleavage.
48
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
a
Table 1. Stabilities of the Conjugates
oligonucleotide
C50 (μM)
TFO
0.18 0.02
0.16 0.03
0.29 0.05
0.30 0.07
0.39 0.05
0.30 0.04
0.26 0.03
TFO-10
TFO-11
TFO-12
TFO-13
TFO-14
TFO-15
a
C50 is the concentration at which 50% of the triplex is formed, after
incubation for 24 h at 37 °C in 50 mM HEPES (pH 7.2), 10 mM
MgCl2, 50 mM NaCl, 10% sucrose, and 0.5 μg/μL tRNA.
three modified nucleobases containing an aminophenyl-thiazole
moiety (1, 4, and 7), conjugated them to a 16-mer TFO that
binds to a specific oligopyrimidine·oligopurine DNA sequence,
and studied their ability to inhibit topo I. Each modified
nucleobase was linked to the TFO with two different spacers
bearing four and six carbon atoms so that we could study the
influence of linker length on topo I inhibition. The six con-
jugates thus obtained (TFO-10−TFO-15) resulted in efficient
topo IB inhibitors, confirming our proposed hypothesis. For
the efficacy of inhibition of the topo IB, it is important to
position and keep the inhibitor in the cleavage complex; the
compounds not conjugated are inactive. Clearly, the TFO plays
the role of positioning the base pair mimic. The observed
cleavage site is a TpT site (Figure 1), suggesting that the nucleo-
bases intercalate between two TA base pairs as hypothesized. In
fact, here we have chosen S analogues that are known to
specifically interact with AT-rich sequences of the DNA16,22
because the targeted topo IB cleavage site at the 3′ end of the
triplex site used in this study contained a T at position −1 and a
T at position +1. In agreement, the DNase I footprinting experi-
ments showed an exaltation of the cleavage in the presence of
the conjugates at the 3′ end where the nucleobases are attached
(Figure 2, lanes 20−25). Furthermore, FID experiments
(Figure 3A,B) showed that the compounds bind to DNA only
when the triplex is formed, suggesting a preference of inter-
calation in the triplex or at the duplex−triplex junction. Impor-
tantly, other intercalators will not act in the same manner because
they may have a certain and different sequence preference.
The three compounds are equally active once conjugated.
This is in perfect agreement with the proposed hypothesis.
First, the three nucleobases differ little chemically: in com-
pounds 1 and 4, the site of attachment to the TFO changes
from the meta to the para position, while compound 7 presents
an additional aromatic substituent. Indeed, conjugate TFO-15
(from nucleobase 7) showed the best activity probably because
the presence of the additional aromatic cycle strengthens the
interactions favoring the intercalation. Second, the TFO posi-
tions the three base pair mimics with the same efficacy in the
topo IB-mediated DNA cleavage complex, and it is this physical
positioning of the base pair mimic as DNA interacalator in the
cleavage complex (as confirmed by the DNase I footprinting
and FID experiments) that is responsible for blocking the
religation reaction of the enzyme and thus the stabilization of
DNA cleavage. The base pair mimics alone have no effect.
Third, the cleavage specificity is dictated by the recognition of
the DNA by the TFO, and all conjugates bear the same TFO.
The efficacy of cleavage of these new conjugates is comparable
to that of the TFO−CPT conjugates developed so far.
Figure 3. Binding of the unconjugated compounds, followed by fluo-
rescence displacement assay and DNase I footprinting. (A) Percentage
of fluorescence obtained after intercalation of BET in double-stranded
DNA (dsDNA) in the presence of increasing concentrations of
compound 14 in the presence (●) or absence (○) of TFO. (B) Per-
centage of fluorescence obtained after intercalation of BET in dsDNA
in the presence of TFO and increasing concentrations of compounds
10−15. (C) Differential cleavage plots comparing susceptibility of the
labeled DNA fragment to DNase I cutting in the presence of com-
pound 14 at increasing concentrations (0.1, 0.5, 1, 2, 10, 50, 100, and
500 μM). Negative values correspond to a ligand-protected site, and
positive values represent enhanced cleavage. Vertical scales are in units
of ln(fa) − ln(fc), where fa is the fractional cleavage at any bond in the
presence of the ligand and fc is the fractional cleavage of the same
bond in the control given closely similar extents of overall digestion.
Each line drawn represents a three-bond running average of individual
data points, calculated by averaging the value of ln(fa) − ln(fc) at any
bond with those of its nearest neighbors. Only a zoom of the region of
the triplex site analyzed by densitometry is shown.
On the basis of these previous results, we hypothesized that
base pair mimics could act as campthotecin analogues, when
positioned and kept in the cleavage complex by triplex forma-
tion, and inhibit the religation reaction after the formation of
topo I−DNA cleavage complex. Accordingly, the conjugation of
the base pair mimic to a TFO is essential. We thus synthesized
49
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
An advantage compared to CPT conjugates8 is that com-
pounds 1, 4, and 7 did not destabilize greatly the triple-helical
structure once conjugated to the TFO (Table 1). Only a slight
destabilization (maximum of 2-fold) was observed. This is in
agreement with previous findings observing that conjugation of
intercalators at the 5′ end increased triplex stability,31−35 but
not at the 3′ end as shown here for the nucleobase conjugates.
In conclusion, on the basis of the crystal structure of the
ternary complex, we have designed and synthesized six new in-
hibitors of topo IB that are very efficient and, even more
importantly, sequence-specific. As we had demonstrated in the
past for campthotecin analogues conjugated to a TFO, these
inhibitors should not present secondary effects because the un-
conjugated compounds are not active. In addition, the com-
pounds do not destabilize triplex formation, a main difference
compared to CPT conjugates. These important features represent
a great advantage for the future development of these new com-
pounds as topo I inhibitors.
P. B. (2010) Sequence-specific targeting of IGF-I and IGF-IR genes by
camptothecins. FASEB J. 24, 2235−2244.
(9) Arimondo, P. B., Bailly, C., Boutorine, A., Ryabinin, V., Syniakov, A.,
́ ̀
Sun, J. S., Garestier, T., and Helene, C. (2001) Directing Topoisomerase
I-mediated DNA Cleavage to Specific Sites by Camptothecin tethered to
Minor and Major Groove Ligands. Angew. Chem., Int. Ed. 40, 3045−
3048.
(10) Arimondo, P. B., Bailly, C., Boutorine, A., Sun, J. S., Garestier, T., and
Helene, C. (1999) Targeting topoisomerase I cleavage to specific
́ ̀
sequences of DNA by triple helix-forming oligonucleotide conjugates.
A comparison between a rebeccamycin derivative and camptothecin.
C. R. Acad. Sci., Ser. III 322, 785−790.
(11) Arimondo, P. B., Boutorine, A., Baldeyrou, B., Bailly, C.,
́ ̀
Kuwahara, M., Hecht, S. M., Sun, J. S., Garestier, T., and Helene, C.
(2002) Design and optimization of camptothecin conjugates of triple
helix- forming oligonucleotides for sequence-specific DNA cleavage by
topoisomerase I. J. Biol. Chem. 277, 3132−3140.
(12) Arimondo, P. B., Thomas, C. J., Oussedik, K., Baldeyrou, B.,
Mahieu, C., Halby, L., Guianvarc’h, D., Lansiaux, A., Hecht, S. M.,
Bailly, C., and Giovannangeli, C. (2006) Exploring the cellular activity
of camptothecin-triple-helix-forming oligonucleotide conjugates. Mol.
Cell. Biol. 26, 324−333.
ASSOCIATED CONTENT
■
(13) Duca, M., Guianvarc’h, D., Oussedik, K., Halby, L., Garbesi, A.,
Dauzonne, D., Monneret, C., Osheroff, N., Giovannangeli, C., and
Arimondo, P. B. (2006) Molecular basis of the targeting of topoisomerase
II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-
forming oligonucleotides. Nucleic Acids Res. 34, 1900−1911.
(14) Arimondo, P. B., Laco, G. S., Thomas, C. J., Halby, L., Pez, D.,
Schmitt, P., Boutorine, A., Garestier, T., Pommier, Y., Hecht, S. M.,
Sun, J. S., and Bailly, C. (2005) Activation of camptothecin derivatives
by conjugation to triple helix-forming oligonucleotides. Biochemistry
44, 4171−4180.
(15) Guianvarc’h, D., Fourrey, J. L., Maurisse, R., Sun, J. S., and Benhida,
R. (2002) Synthesis, incorporation into triplex-forming oligonucleotide,
and binding properties of a novel 2′-deoxy-C-nucleoside featuring a 6-
(thiazolyl-5)benzimidazole nucleobase. Org. Lett. 4, 4209−4212.
(16) Guianvarc’h, D., Fourrey, J. L., Maurisse, R., Sun, J. S., and
Benhida, R. (2003) Design of artificial nucleobases for the recognition
of the AT inversion by triple-helix forming oligonucleotides: A
structure-stability relationship study and neighbour bases effect. Bioorg.
Med. Chem. 11, 2751−2759.
(17) Li, J. S., Chen, F. X., Shikiya, R., Marky, L. A., and Gold, B.
(2005) Molecular recognition via triplex formation of mixed purine/
pyrimidine DNA sequences using oligoTRIPs. J. Am. Chem. Soc. 127,
12657−12665.
(18) Mizuta, M., Banba, J., Kanamori, T., Tawarada, R., Ohkubo, A.,
Sekine, M., and Seio, K. (2008) New nucleotide pairs for stable DNA
triplexes stabilized by stacking interaction. J. Am. Chem. Soc. 130,
9622−9623.
(19) Rahman, S. M., Seki, S., Obika, S., Haitani, S., Miyashita, K., and
Imanishi, T. (2007) Highly stable pyrimidine-motif triplex formation
at physiological pH values by a bridged nucleic acid analogue. Angew.
Chem., Int. Ed. 46, 4306−4309.
(20) Rusling, D. A., Powers, V. E., Ranasinghe, R. T., Wang, Y.,
Osborne, S. D., Brown, T., and Fox, K. R. (2005) Four base
recognition by triplex-forming oligonucleotides at physiological pH.
Nucleic Acids Res. 33, 3025−3032.
(21) Sasaki, S., Taniguchi, Y., Takahashi, R., Senko, Y., Kodama, K.,
Nagatsugi, F., and Maeda, M. (2004) Selective formation of stable
triplexes including a TA or a CG interrupting site with new bicyclic
nucleoside analogues (WNA). J. Am. Chem. Soc. 126, 516−528.
(22) Guianvarc’h, D., Benhida, R., Fourrey, J. L., Maurisse, R., and
Sun, J. S. (2001) Incorporation of a novel nucleobase allows stable
oligonucleotide-directed triple helix formation at the target sequence
containing a purine·pyrimidine interruption. Chem. Commun.,, 1814−
1815.
S
* Supporting Information
1H NMR and 13C NMR spectra as well as HRMS spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: paola.arimondo@etac.cnrs.fr. Phone: +33534506492.
Fax: +33534503492. Present address: USR CNRS-PierreFabre n°3388
ETaC, CRDPF, 3 avenue H. Curien, 31100 Toulouse, France.
Author Contributions
P.V. and M.D. contributed equally to this work.
Funding
This work was supported by grants to P.V. from Association
pour la Recherche sur le Cancer (ARC).
ACKNOWLEDGMENTS
We thank Lionel Dubost and Arul Marie of the mass
spectrometry facility of the MNHN.
■
REFERENCES
■
(1) Champoux, J. J. (2001) DNA Topoisomerases: Structure,
Function and Mechanism. Annu. Rev. Biochem. 70, 369−413.
(2) Wang, J. C. (2002) Cellular roles of DNA topoisomerases: A
molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430−440.
(3) Pommier, Y. (2006) Topoisomerase I inhibitors: Camptothecins
and beyond. Nat. Rev. Cancer 6, 789−802.
(4) Hande, K. R. (2003) Topoisomerase II inhibitors. Cancer
Chemother. Biol. Response Modif. 21, 103−125.
(5) Staker, B. L., Hjerrild, K., Feese, M. D., Behnke, C. A., Burgin,
A. B. Jr., and Stewart, L. (2002) The mechanism of topoisomerase I
poisoning by a camptothecin analog. Proc. Natl. Acad. Sci. U.S.A. 99,
15387−15392.
(6) Staker, B. L., Feese, M. D., Cushman, M., Pommier, Y.,
Zembower, D., Stewart, L., and Burgin, A. B. (2005) Structures of
three classes of anticancer agents bound to the human topoisomerase
I-DNA covalent complex. J. Med. Chem. 48, 2336−2345.
(7) Marchand, C., Antony, S., Kohn, K. W., Cushman, M., Ioanoviciu,
A., Staker, B. L., Burgin, A. B., Stewart, L., and Pommier, Y. (2006) A
novel norindenoisoquinoline structure reveals a common interfacial
inhibitor paradigm for ternary trapping of the topoisomerase I-DNA
covalent complex. Mol. Cancer Ther. 5, 287−295.
(23) Vekhoff, P., Halby, L., Oussedik, K., Dallavalle, S., Merlini, L.,
Mahieu, C., Lansiaux, A., Bailly, C., Boutorine, A., Pisano, C., Giannini,
G., Alloatti, D., and Arimondo, P. B. (2009) Optimized synthesis and
(8) Oussedik, K., Francois, J. C., Halby, L., Senamaud-Beaufort, C.,
Toutirais, G., Dallavalle, S., Pommier, Y., Pisano, C., and Arimondo,
50
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51

Biochemistry
Article
enhanced efficacy of novel triplex-forming camptothecin derivatives
based on gimatecan. Bioconjugate Chem. 20, 666−672.
(24) Shim, Y. H., Arimondo, P. B., Laigle, A., Garbesi, A., and Lavielle, S.
(2004) Relative DNA binding affinity of helix 3 homeodomain analogues,
major groove binders, can be rapidly screened by displacement of
prebound ethidium bromide. A comparative study. Org. Biomol. Chem. 2,
915−921.
(25) Wang, Z. (2010) Hantzsch Thiazole Synthesis. In Comprehensive
Organic Name Reactions and Reagents, pp 1330−1334, John Wiley &
Sons, New York.
(26) Arimondo, P. B., Angenault, S., Halby, L., Boutorine, A.,
Schmidt, F., Monneret, C., Garestier, T., Sun, J. S., Bailly, C., and
́ ̀
Helene, C. (2003) Spatial organization of topoisomerase I-mediated
DNA cleavage induced by camptothecin-oligonucleotide conjugates.
Nucleic Acids Res. 31, 4031−4040.
(27) Lacroix, L., Lacoste, J., Reddoch, J. F., Mergny, J. L., Levy, D. D.,
Seidman, M. M., Matteucci, M. D., and Glazer, P. M. (1999) Triplex
formation by oligonucleotides containing 5-(1-propynyl)-2′-deoxyuridine:
Decreased magnesium dependence and improved intracellular gene
targeting. Biochemistry 38, 1893−1901.
́ ̀
(28) Chomilier, J., Sun, J. S., Collier, D. A., Garestier, T., Helene, C.,
and Lavery, R. (1992) A computational and experimental study of the
bending induced at a double-triple helix junction. Biophys. Chem. 45,
143−152.
(29) Arimondo, P. B., Bailly, C., Boutorine, A., Moreau, P., Prudhomme,
́ ̀
M., Sun, J. S., Garestier, T., and Helene, C. (2001) Triple helix-forming
oligonucleotides conjugated to indolocarbazole poisons direct
topoisomerase I-mediated DNA cleavage to a specific site. Bioconjugate
Chem. 12, 501−509.
(30) Arimondo, P. B., Bailly, C., Boutorine, A., Prudhomme, M., Sun,
J.-S., Garestier, T., and Helene, C. (2000) Recognition and Cleavage of
́ ̀
DNA by Rebeccamycin- or Benzopyridoquinoxaline-Triplex-Forming
Oligonucleotide Conjugates. Bioorg. Med. Chem. 8, 777−784.
(31) Capobianco, M. L., De Champdore, M., Francini, L., Lena, S.,
Garbesi, A., and Arcamone, F. (2001) New TFO conjugates
containing a carminomycinone-derived chromophore. Bioconjugate
Chem. 12, 523−528.
(32) Garbesi, A., Bonazzi, S., Zanella, S., Capobianco, M. L., Giannini,
G., and Arcamone, F. (1997) Synthesis and binding properties of
conjugates between oligodeoxynucleotides and daunorubicin
derivatives. Nucleic Acids Res. 25, 2121−2128.
(33) Stierle, V., Duca, M., Halby, L., Senamaud-Beaufort, C.,
Capobianco, M. L., Laigle, A., Jolles, B., and Arimondo, P. B. (2008)
Targeting MDR1 gene: Synthesis and cellular study of modified
daunomycin-triplex-forming oligonucleotide conjugates able to inhibit
gene expression in resistant cell lines. Mol. Pharmacol. 73, 1568−1577.
(34) Sun, J. S., Franco
T., Roig, V., Chassignol, M., Thuong, N. T., and Hel
̧
is, J. C., Montenay-Garestier, T., Saison-Behmoaras,
ene, C. (1989)
́
̀
Sequence-specific intercalating agents. Intercalation at specific sequences on
duplex DNA via major-groove recognition by oligonucleotide intercalator
conjugates. Proc. Natl. Acad. Sci. U.S.A. 86, 9198−9202.
(35) Sun, J. S., Giovannangeli, C., Francois, J. C., Kurfurst, R.,
Montenay-Garestier, T., Asseline, U., Saison-Behmoaras, T., Thuong,
́ ̀
N. T., and Helene, C. (1991) Triple-helix formation by α oligo-
deoxynucleotides and α oligodeoxynucleotide-intercalator conjugates.
Proc. Natl. Acad. Sci. U.S.A. 88, 6023−6027.
51
dx.doi.org/10.1021/bi2012959 | Biochemistry 2012, 51, 43−51