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J. Biol. Chem., Vol. 277, Issue 5, 3132-3140, February 1, 2002
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§,,
,,,, and
From the Laboratoire de Biophysique, UMR 8646 CNRS,
Muséum National d'Histoire Naturelle, INSERM U201, 43 rue
Cuvier, 75231 Paris cedex 05, France, ¶ INSERM U524 and
Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret,
Institut de Recherches sur le Cancer de Lille, Place Verdun,
59045 Lille, France, and the Department of Chemistry, University
of Virginia, Charlottesville, Virginia 22901
Received for publication, October 23, 2001, and in revised form, November 26, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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To achieve a sequence-specific DNA cleavage by
topoisomerase I, derivatives of the antitumor drug camptothecin
have been covalently linked to triple helix-forming oligonucleotides
that bind in a sequence-specific manner to the major groove of
double-helical DNA. Triplex formation at the target sequence positions
the drug selectively at the triplex site, thereby stimulating
topoisomerase I-mediated DNA cleavage at this site. In a
continuous effort to optimize this strategy, a broad set of conjugates
consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers
of variable length, and (iii) camptothecin derivatives substituted on
the A or B quinoline ring were designed and synthesized. Analysis of
the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of
both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced
strong and highly specific cleavage at a site adjacent to the triplex.
Furthermore, the drug-stabilized DNA-topoisomerase I cleavage
complexes were shown to be more resistant to salt-induced reversal than
the complexes induced by camptothecin alone. Such rationally designed
camptothecin conjugates could provide useful antitumor drugs directed
selectively against genes bearing the targeted triplex binding site. In
addition, they represent a powerful tool to probe the molecular
interactions in the DNA-topoisomerase I complex.
The reaction between double-stranded DNA and topoisomerase I
produces a covalent 3'-phosphorotyrosyl adduct, usually referred to as
the cleavage complex (1, 2). Under physiological conditions, the
covalent intermediate is barely detectable, because a fast religation
step occurs after relaxation of the DNA constraints. A number of drugs,
such as the antitumor alkaloid camptothecin (CPT),1 can convert
topoisomerase I into a cell poison by blocking the religation step,
thereby enhancing the formation of persistent DNA breaks responsible
for cell death (1, 3, 4). However, topoisomerase I poisons display a
weak sequence specificity. Mainly one or two nucleotides on the 3' and
5'-side of the cleavage site (essentially thymine-guanine steps
in the case of CPT) represent the only recognition elements. As a
result, drugs like CPT induce massive nonspecific DNA damage in cells
and can affect any gene within the genome. The identification, over the
last decade, of genes that play a key role in the progression and
maintenance of a specific disease, such as oncogenes and tumor
suppressor, calls for the development of drugs able to regulate the
expression and functions of these genes. For this reason, the design of
molecules that bind to specific sequences in DNA is urgently needed. To direct the topoisomerase I enzymatic reaction to particular sites, topoisomerase I poisons, including camptothecin and rebeccamycin derivatives, have been covalently attached to sequence-specific DNA
ligands, such as triplex-forming oligonucleotides (TFO) (5-7) and
hairpin polyamides (8, 9) that bind in a sequence-specific manner to
the major and minor groove of double-helical DNA, respectively.
Interesting results have been obtained previously with triplex-forming
oligonucleotide-camptothecin conjugates (TFO-CPT) (5, 6), but from
initial attempts to direct topoisomerase I-mediated cleavage to a
specific site, it was clear that the system had to be carefully
optimized to enhance the specificity and efficacy of cleavage (7). We
report here on the effect of the length of the triple-helical structure
and the linker arm between the TFO and the poison, as well as the
influence of the drug orientation with respect to the topoisomerase I
cleavage site. The DNA binding and topoisomerase I-mediated cleaving
properties of the conjugates were investigated by PAGE at
nucleotide resolution, and their effects on the stability of the
topoisomerase I-DNA complexes were also investigated. Altogether the
results attest unequivocally that the recognition and cleavage of DNA
by topoisomerase I can be fully controlled by the attachment of a
CPT-type poison to a TFO, but the structure of the hybrid molecule must
be precisely adapted to direct the cleavage reaction to the target site.
Oligonucleotides and DNA Fragment
Oligonucleotides were purchased from Eurogentec and purified
using quick spin columns and Sephadex G-25 fine (Roche Molecular Biochemicals). The concentrations were determined
spectrophotometrically at 25 °C using molar extinction coefficients
at 260 nm calculated from a nearest neighbor model (10).
The nomenclature of the oligonucleotides and conjugates is as follows.
The abbreviation TFO is followed by a number referring to the length of
the oligonucleotide, followed by the letter L (for linker) and the
number of carbon atoms in the linker, and finally, by the denomination
of the camptothecin derivative (10CPT for 10-carboxycamptothecin or
7CPT for 7-(2-aminoethyl)camptothecin). For example, TFO20-L4-10CPT
stands for the 20-mer TFO linked through the diaminobutyl spacer to
10-carboxycamptothecin.
The plasmid pBSK(+/ Topoisomerase Poisons
All the drugs were dissolved in dimethyl sulfoxide at 3 mg/ml
and then diluted further with water. The final dimethyl sulfoxide concentration never exceeded 0.3% (v/v) in all assays.
10-Carboxycamptothecin (kindly provided by Dr. Mark Matteucci,
Gilead Sciences; structure in Fig. 2) was conjugated to the
terminal amino group of a diaminoalkyl linker arm at the 3'-end of the
oligonucleotide as described in Ref. 6. The linker arm was attached by
reaction of the corresponding alkyldiamine to the 3'-phosphorylated
oligonucleotide activated by treatment with
N-methylimidazole, dipyridyl disulfide, and triphenylphosphine as described in Ref. 12.
7-(2-Aminoethyl)camptothecin
Synthesis 7-(2-Hydroxyethyl)camptothecin (2)--
To a
suspension of 100 mg (0.29 mmol) of camptothecin and 160 mg (0.58 mmol)
of FeSO4·7 H2O in 2 ml of ethanol and 4 ml of H2O was added dropwise 2 ml of
H2SO4. This suspension was cooled to 0 °C
and then stirred and treated dropwise with 1 ml of 30% H2O2. The reaction mixture was stirred at room
temperature for 3 h and then diluted with water, which afforded a
precipitate. The precipitate, which consisted mostly of CPT and
7-methyl CPT in addition to the desired product, was triturated with
10% EtOH in CHCl3, and the combined extract was
concentrated to afford the crude product. This material was purified by
chromatography on silica gel; elution was effected with
MeOH-CHCl3 mixtures (1:99 MeOH-CHCl3 7-(2-p-Toluenesulfoxyethyl)camptothecin (3)--
A
solution containing 164 mg (0.42 mmol) of
7-(2-hydroxyethyl)camptothecin (2) in 2 ml of pyridine at
4 °C was treated with 120 mg (0.63 mmol) of recrystallized
p-toluenesulfonyl chloride. The reaction mixture was stirred
under argon at 4 °C for 40 h and then concentrated under
diminished pressure. The residue was dissolved in CHCl3,
and the organic phase was washed successively with 0.8 M
aqueous citric acid and water and then dried
(Na2SO4) and concentrated under diminished
pressure. The crude product was purified on a silica gel column (8 × 1.3 cm); elution was carried out with 1% MeOH in CHCl3.
This afforded 7-(2-p-toluenesulfoxyethyl)CPT (3)
as a pale yellow powder, contaminated with a small amount of
7-(2-chloroethyl)CPT: yield, 214 mg (94%); silica gel TLC
Rf = 0.29 (7:3 CHCl3-acetone);
1H NMR (CDCl3) 7-(2-Azidoethyl)camptothecin (4)--
A solution
containing 214 mg (0.39 mmol) of
7-(2-p-toluenesulfoxyethyl)CPT (3) in 2 ml of
N,N-dimethylformamide was treated with 254 mg (3.91 mmol) of sodium azide. The reaction mixture was stirred under
argon at room temperature for 24 h and then concentrated under
diminished pressure. The residue was suspended in CHCl3,
filtered to remove unreacted sodium azide, and then concentrated under
diminished pressure. The residue was purified by chromatography on a
silica gel column (8 × 1.3 cm); elution was carried out with
CHCl3-acetone mixtures (1% 7-(2-Aminoethyl)camptothecin 7CPT (5)--
A
solution containing 141 mg (0.34 mmol) of 7-(2-azidoethyl)CPT
(4) and 94 mg of 10% palladium-on-carbon in 28 ml of EtOH
and 1.1 ml of concentrated HCl was stirred under a H2
atmosphere at room temperature for 20 h. The reaction mixture was
filtered through Celite to remove the catalyst, and then the filtrate
was washed with CHCl3 to remove the by-product 7-ethylCPT,
and then the aqueous solution was concentrated to dryness under
diminished pressure. The residue was purified by chromatography on a
silica gel column (8 × 1.3 cm); elution was carried out with
MeOH-CHCl3 mixtures (1% Conjugation to the TFOs--
150 µg of 3'-phosphorylated
oligonucleotides, TFO16 and TFO18 (see Fig. 2), were conjugated to the
7CPT via Topoisomerase I Cleavage Assays
The radiolabeled 324-bp target duplex (50 nM) was
incubated for 1 h at 30 °C, in 50 mM Tris-HCl, pH
7.5, 60 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM EDTA, and 30 µg of
bovine serum albumin, in the presence of the TFO (at the indicated
concentration) to form the triplex (total reaction volume, 20 µl). To
analyze the topoisomerase I DNA cleavage products, 10 units of enzyme (Invitrogen) were added to the duplex, preincubated as described above
with either the TFO or/and the drugs, and incubated for 30 min at
30 °C. The DNA-topoisomerase I cleavage complexes were dissociated
by the addition of SDS (final concentration, 0.25%) and of proteinase
K (Sigma) to 250 µg/ml, followed by incubation for 35 min at
56 °C. After ethanol precipitation, all of the samples were
resuspended in 6 µl of formamide, heated at 90 °C for 4 min, and
then chilled on ice for 4 min before being loaded onto a denaturing 8%
polyacrylamide gel (19:1 acrylamide:bisacrylamide) containing 7.5 M urea in 1× TBE buffer (50 mM Tris base, 55 mM boric acid, 1 mM EDTA). To quantitate the
extent of cleavage, the gels were scanned with a Molecular Dynamics
445SI PhosphorImager. For the determination of cleavage levels,
normalization relative to total loading was performed. To investigate
the reversal of the drug-induced cleavage complexes, after incubation
with topoisomerase I and prior addition of SDS and proteinase K,
increasing concentrations of NaCl were added as indicated for 5 min.
The samples were then processed as described above. The bases of the 3'
and 5' termini of the analyzed cleavage sites were numbered DNase I Protection Assays
The DNA template for the DNase I protection assay on the
oligopyrimidine-containing strand of the target was obtained as
described above. 1 µl of DNase I (final concentration, 0.2 mg/ml;
Sigma) diluted in 1 mM MgCl2, 1 mM
MnCl2, and 20 mM NaCl was added to the
radiolabeled duplex and preincubated for 1 h at 30 °C in the buffer described above in the absence or presence of TFO. The reaction
and analysis were performed as described in Ref. 7.
The synthesis of 7-(2-aminoethyl)CPT (7CPT), a new CPT derivative
required for the preparation of TFO-CPT conjugates attached to the
alkaloid via the 7-position, was carried out as outlined in Fig.
1 starting from camptothecin itself. Thus
treatment of CPT (1) with FeSO4,
H2O2, and H2SO4 in
aqueous EtOH afforded 7-(2-hydroxyethyl)CPT (2), as
described previously (13). Following activation of the primary OH group
as a tosylate, treatment with sodium azide in
N,N-dimethylformamide afforded
7-(2-azidoethyl)CPT (4) in 81% overall yield from
hydroxyethylCPT derivative 2. Hydrogenolysis then provided
the 7-(2-aminoethyl)camptothecin (7CPT (5)).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) was bought from Promega, and the 77-bp
target duplex was inserted between the BamHI and
EcoRI sites. The digestion of the plasmid by
PvuII and EcoRI yielded a 324-mer fragment
suitable for 3'-end labeling by the Klenow polymerase and
[
-32P]dATP (Amersham Biosciences, Inc.). The detailed
procedures for isolation, purification, and labeling of this duplex DNA
fragment have been previously described (11).
7-(2-Aminoethyl)camptothecin 7CPT (5)
was obtained by synthetic transformation of the natural product
camptothecin (1) as outlined in Fig. 1.
3:97
MeOH-CHCl3). The desired 7-(2-hydroxyethyl)CPT (2) crystallized from ethanol as a colorless
microcrystalline solid: yield, 19 mg (17%); silica gel TLC
Rf = 0.09 (7:3 CHCl3-acetone); mass
spectrum (chemical ionization), m/z 393 (M+H)+.
1.04 (t, 3H), 1.90 (m, 2H),
2. 28 (s, 3H), 3.48 (t, 2H), 3.87 (br, 1H), 4.44 (t, 2H), 5.15 (s, 2H),
5.52 (AB quartet, 2H), 6.95 (d, 2H), 7.32 (d, 2H), 7.62 (t, 1H), 7.65 (s, 1H), 7.79 (t, 1H), 7.89 (d, 1H), and 8.20 (d, 1H); 13C
NMR (CDCl3) 7.8, 21.6, 29.4, 31.6, 49.7, 66.3, 68.2, 72.7, 98.1, 118.9, 122.8, 126.7, 127.2, 128.2, 128.9, 129.5, 130.3, 130.7, 131.9, 138.0, 145.0, 146.4, 149.1, 150.1, 151.6, 157.4, and
173.8; mass spectrum (chemical ionization), m/z 347.2 (M+H)+.
3% acetone in CHCl3). This afforded 7-(2-azidoethyl)CPT (4)
contaminated with a small amount of 7-vinylcamptothecin: yield 141 mg
(86%); silica gel TLC Rf = 0.35 (7:3
CHCl3-acetone); 1H NMR (CDCl3) 1.02 (t, 3H), 1.88 (m, 2H), 3.41 (m, 2H), 3.77 (br, 1H), 3.82 (t, 2H),
5.34 (s, 2H), 5.52 (AB quartet, 2H), 7.67 (s, 1H), 7.69 (t, 1H), 7.82 (t, 1H), 8.04 (d, 1H), and 8.26 (d, 1H); 13C NMR
(CDCl3) 7.8, 29.6, 31.6, 50.0, 50.8, 66.4, 72.7, 98.4, 118.8, 122.9, 126.9, 128.2, 128.9, 130.4, 130.9, 139.9, 146.6, 149.2, 150.1, 151.8, 157.6, and 173.9; mass spectrum (chemical ionization),
m/z 418 (M+H)+.
40% MeOH in
CHCl3) to afford 7-(2-aminoethyl)CPT (5) as a
pale yellow powder: yield 50 mg (38%); silica gel TLC
Rf = 0.11 (1:1 CHCl3-MeOH);
1H NMR (Me2SO-d6) 0.86 (t, 3H), 1.86 (m, 2H), 3.15 (m, 2H), 3.60 (m, 2H), 5.39 (s, 2H),
5.44 (s, 2H), 6.55 (br, 1H), 7.34 (s, 1H), 7.76 (t, 1H), 7.88 (t, 1H),
8.19 (d, 1H), 8.43 (d, 1H), and 8.49 (br, 2H); 13C NMR
(Me2SO-d6) 7.8, 27.1, 30.3, 38.1, 50.0, 65.3, 72.4, 96.8, 119.1, 124.1, 126.9, 128.0, 129.9, 130.0, 130.2, 139.1, 145.9, 148.5, 150.1, 152.1, 156.8, and 172.5; mass
spectrum (chemical ionization), m/z 392 (M+H)+.
-aminocaproic acid linker according to the methods
described in Ref. 12. After precipitation as hexadecyltrimethylammonium
salt, the oligonucleotide was then dissolved in 50 µl of dry
Me2SO. Methylaminopyridine (5 µg) and dipyridyl disulfide
and triphenylphosphine solutions (25 µl each 1.2 M in
Me2SO) were added. After 15 min of incubation at room
temperature, the activated oligonucleotide was precipitated with 2%
LiClO4 in acetone, rinsed with acetone, and dissolved in 50 µl of aqueous solution of -aminocaproic acid (6 mg) with 5 µl of
triethylamine. After 2 h of incubation the oligonucleotide was
precipitated with LiClO4 in acetone, followed by
precipitation with ethanol. The terminal carboxyl group of the linker
attached to the 3'-end of the oligonucleotides was again activated with dipyridyl disulfide/triphenylphosphine as described above and then
reacted with the NH2 group of the ethyl chain of the 7CPT (see Fig. 2). After precipitation with LiClO4/acetone, the
conjugate was purified by reverse phase HPLC using a linear
acetonitrile gradient (0-80% CH3CN in 0.2 M
(NH4)OAc). The average yield was 60%. The
oligonucleotide-drug conjugates were characterized by UV spectroscopy.
1 and +1, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Synthetic route used for the
preparation of 7-(2-aminoethyl)camptothecin 7CPT.
The target DNA sequence used in this study and the newly
designed TFO-CPT conjugates are shown in Fig.
2. The 77-bp target duplex, containing a
central 23-bp oligopyrimidine· oligopurine target sequence for
triplex formation, was inserted between the BamHI and
EcoRI sites of plasmid pBSK(+/), and a resulting 324-mer 3'-end 32P-radiolabeled fragment was used for topoisomerase
I DNA cleavage and DNA binding assays. We have used this system
previously to study triplex formation with 16-nucleotide-long oligomers
(underlined and bold in Fig. 2) (6, 7, 14). The
TFO moiety of the conjugates consists of 16-20-nucleotide-long
oligomers (TFO) containing 5-methyldeoxycytosines and
5-propynyldeoxyuracils to increase triplex stability (15). The spacer
corresponds to an alkyl linker containing 4-10 methylene units. The
site of attachment of the drug was also varied by the use of
camptothecin derivatives substituted on either the A or the B ring of
the quinoline moiety; for comparison, 10-carboxycamptothecin and
7-aminoethylcamptothecin derivatives were attached to the TFO via hexyl
linkers.
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Effect of the Triplex Size and Its Positioning with Respect to
Topoisomerase I Cleavage Sites--
We investigated how the increase
in length of the TFO by two nucleotides on the 3'-side would affect
topoisomerase I-mediated DNA cleavage in the proximity of the triplex
site by using the previously described 16-mer, TFO16 (5, 6); an 18-mer,
TFO18; and a 20-mer, TFO20 (Fig. 2). Even though the potential triplex site contains a 23-bp oligopyrimidine·oligopurine sequence, we limited our study to the 20-mer because a longer oligonucleotide would
overlap the strong CPT-induced cleavage site b2 determined previously (6). In a first set of experiments, the 324-bp restriction fragment labeled at the 3'-end of the oligopyrimidine-containing strand
was incubated with the TFOs and subjected to limited DNase I cleavage
(Fig. 3). As expected, the DNase I
cleavage is strongly inhibited at the target
oligopyrimidine·oligopurine tract, and the footprint is clearly
extended with the 18- and 20-mer TFOs (lanes 12 and
13) compared with the 16-mer (lane 11). In
parallel experiments, the duplex DNA target was incubated with the 16-, 18-, and 20-mer TFOs prior to cleavage by topoisomerase I (Fig. 3). The reactions were performed in the presence (lanes
4-6) or absence (lanes 7-9) of camptothecin. In the
presence of 10-carboxycamptothecin alone (lane 3), strong
topoisomerase I-mediated cleavage sites could be detected both on the
3'-side of the triplex region (at b1 and b2
sites, at 4 and 7 bp from the 16-mer triplex termini, respectively) and
on the 5'-side of the triplex region (site a, at 8-bp from
the triple helix end). Additional sites marked c, d, e, and f were also detected. The
presence of the 16-mer TFO did not greatly affect the 10CPT-stimulated
cleavage (lane 4); only a decrease in cleavage at site
a was detected. As the length of the triplex increased on
the 3'-end (lanes 5 and 6), cleavage at sites
b1 and b2 gradually decreased, whereas cleavages
at sites a and c-f were not significantly
altered. A new and weak cleavage site, designated b3,
appeared in the presence of the 20-mer TFO (lane 6). A small
effect was observed in the presence of the triple helix alone
(lanes 7-9), but again, in the presence of the 20-mer TFO,
the weak site b3 appeared, and cleavage at site
b1 was abolished.
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Optimization of the Linker Arm-- An important parameter that must be adjusted to optimize topoisomerase I-induced cleavage at the 3'-end of the triple helix is the length of the linker arm between the oligonucleotide and camptothecin. To evaluate this parameter, 10CPT was attached to the TFO16 either through a diaminobutyl (TFO16-L4-10CPT), a diaminohexyl (TFO16-L6-10CPT), or a diaminodecyl linker (TFO16-L10-10CPT) (Fig. 2). The spacer was first attached to the 3'-phosphorylated TFO upon activation by dipyridyl disulfide and triphenylphosphine in the presence of N-methylimidazole, and then the other terminal NH2 group of the linker was reacted with the N-hydroxysuccinimide-activated ester of 10-carboxycamptothecin.
Fig. 4 shows the analysis of the
topoisomerase I cleavage products on the radiolabeled 324-nucleotide
duplex target. All of the conjugates strictly restricted DNA cleavage
by topoisomerase I to the 3'-side of the duplex/triplex junction
(sites b1 and b2). Cleavage at sites a
and c-f completely disappeared. This is a strong indication
that the enzyme has been targeted to a specific region, as expected. As
the length of the spacer increases, the efficacy of cleavage at site
b1 by the TFO16-10CPT conjugate decreases; cleavage is
still pronounced with the L6 conjugate (lane 5) but is much
poorer with the L10 analog (lane 6).
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To delineate further how the presence of the triple helix influences triplex-directed topoisomerase I-mediated DNA cleavage, we then used triplexes of different lengths in combination with different spacers. Because the conjugate containing the longest linker L10 (TFO16-L10-10CPT) is the least potent at inducing cleavage at site b1 and, furthermore, induces some cleavage at site b2 located at 7 bp from the 3' triplex end, we restricted the study to the diaminobutane (L4) and diaminohexane (L6) spacers.
Effect of the Triplex Size and Length of the Linker Arm--
The
capacity of the 16-, 18-, and 20-mer TFO-L4/6-10CPT conjugates to
induce sequence-specific topoisomerase I-mediated DNA cleavage was
compared in Fig. 5. Again DNA cleavage by
topoisomerase I was restricted to the 3'-side of the duplex/triplex
junction (sites b1 and b2). In the 16-mer
conjugates series (lanes 4 and 5), the cleavage
efficiency of topoisomerase I at site b1 decreased as the
length of the linker increased (as discussed above). The reverse
situation was observed in the 18-mer series (lanes 6 and 7). In this case, we detected a much stronger cleavage at
site b1 in the presence of the conjugate containing the L6
linker than the analog containing the L4 linker. However, the 18-mer
TFO18-L6-10CPT conjugate not only recruited topoisomerase I at site
b1 but also promoted cleavage at the adjacent site
b2, whereas the 16-mer conjugates stimulated DNA cleavage
only at site b1. As in the presence of the triple helix
alone (Fig. 3), the 20-mer conjugates strongly reduced the access of
topoisomerase I to site b1, and they stimulated cleavage
only at the adjacent site b2. Of the 20-mer conjugates, the
one containing the hexamethylene linker was the most efficient.
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The recruitment of topoisomerase I to sites b1 or b2 is markedly dependent on the length of the TFO and/or linker. In all cases, cleavage remained specific to the proximity of the 3' triplex end; the figure clearly shows the loss of cleavage at site a and at sites c-f. All conjugates form triple helices, as revealed by footprinting experiments (data not shown).
Influence of the Camptothecin Moiety-- The experiments described above as well as the studies previously reported by us (6) and others (5) were all performed with a CPT derivative substituted on the A ring. Here we extended the study to a B ring-substituted analog. According to the topoisomerase I-DNA-camptothecin ternary complex models (16, 17) and structure-activity relationships studies (18), there is a space for substitutions at positions 7 and 10 on the B and A ring of the quinoline moiety of CPT, respectively, without decrease in activity. Therefore, we synthesized conjugates of the 16- and 18-mer TFO linked to 7-(2-aminoethyl)camptothecin via an hexyl linker (structure in Fig. 2). The DNA recognition and cleaving properties of these conjugates were compared with the ones of the 10-carboxycamptothecin analogs. The differences in intensity of cleavage observed for the four different combinations of TFO16/18 and 7/10CPT provide some structural information regarding the ternary DNA-topoisomerase-poison complex.
Fig. 6 shows that the topoisomerase I
cleavage profile of the free 7CPT differs markedly from the 10CPT one.
Cleavage at site b1 was stronger than at site b2;
the opposite effect was observed with 10CPT (compare lanes 3 and 6). Furthermore, cleavage at site a and
f was weaker with 7CPT than with 10CPT. Noteworthy, the two
TFO-7CPT conjugates induced cleavage only at sites b1 and b2 on the 3'-side of the triple helix. Cleavage at all the
other sites of the drug (a and c-f) was
abolished, attesting that topoisomerase I-mediated DNA cleavage had
been specifically directed by triplex formation. The 16-mer conjugate
of 7CPT (TFO16-L6-7CPT, lane 4) specifically enhanced
cleavage at site b1 (7.6-fold) and reduced it at site
b2 as compared with free 7CPT. The reverse profile was
observed with the 18-mer conjugate TFO18-L6-7CPT (lane 5), which increased cleavage at site b2 by 10-fold and decreased
it at site b1. On the contrary, 10CPT conjugates
(TFO16-L6-10CPT and TFO18-L6-10CPT, lanes 7 and
8, respectively) stimulated cleavage preferentially at site
b1 and only the longer, the 18-mer (lane 8),
cleaved at site b2 to give a profile similar to the one
observed with the 16-mer 7CPT conjugate (lane 4).
|
Concentration Dependence of Topoisomerase I-mediated DNA
Cleavage--
Next we compared the concentration dependence of the
cleavage efficacy of the free drug to that of the TFO-drug conjugates. Fig. 7A shows topoisomerase
I-mediated DNA cleavage in the presence of increasing concentrations of
10CPT (lanes 3-8) and TFO16-L4-10CPT (lanes
9-14) (from 0.01 to 5 µM). It is clear that the
conjugate induces efficient DNA cleavage even at a concentration as low as 0.01 µM. At this concentration 10CPT alone (lane
3) shows little effect compared with topoisomerase I alone
(lane 2). Band intensities were quantified by PhosphorImager
analysis. Fig. 7B compares the percentage of cleavage as a
function of the drug concentration for 10CPT (circles) and
TFO16-L4-10CPT (triangles) at sites d, b2, b1, and a. TFO16-L4-10CPT induces
cleavage at site b1 even at 0.01 µM; in
contrast, at the other sites no cleavage was observed with the
conjugate. Similar experiments were performed with various 16- and
18-mer conjugates of both 10CPT and 7CPT (Fig.
8). Here again the conjugates strongly
enhanced topoisomerase I-mediated cleavage selectively at the triplex
site b1/b2. The tethered molecules are
considerably more potent than the free drug at inducing DNA cleavage.
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|
Salt-induced Reversal of the Cleavage Complex in the Presence of
Untethered and Tethered CPT--
We then examined the reversibility of
the topoisomerase I cleavage complexes induced by the drug alone and by
the drug conjugated to the TFOs. CPT-stabilized cleavage complexes are
rapidly reversible by increasing salt (NaCl) concentrations (19), and
this method can be used to compare the stability of the cleavage
complexes induced by different drugs. In Fig.
9, the effect of 1 µM
10-carboxycamptothecin is compared with that of conjugates
TFO16-L4-10CPT and TFO16-L6-7CPT at 0.5 µM. For each
sample, after topoisomerase I reaction, increasing concentrations of
salt (0, 50, 100, 200, 400, and 600 mM NaCl) were added for
5 min. prior to proteinase K digestion. The results leave no doubt that
the conjugates strongly increased the stability of the cleavage complex
at site b1. High concentrations of NaCl were needed to
reverse the cleavage complexes induced by the conjugated drug compared
with the drug alone.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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In the present study we probed the DNA-topoisomerase I cleavage
complex targeted to a specific site using triple-helical structures of
different lengths and conjugated to two camptothecin derivatives via
spacers of variable size (Fig. 2). We first investigated how the
increase of the length of the triple-helical structure at its 3'-end
modified DNA cleavage by topoisomerase I at sites adjacent to the
triplex site. As the length of the triplex increased at the 3'-end
(TFO16 TFO18 TFO20) (Fig. 3), topoisomerase I cleaved further
away from the triplex end, and a weak new site was observed at 7 bp
from the 3'-end of the 20-mer (site b3).
The TFOs were linked either to 10CPT or 7CPT using for the
former spacers of different length (butyl-, hexyl-, and decyldiamines) between the oligonucleotide and the drug, and for the latter
-aminocaproic acid (Fig. 2). The capacity of the various conjugates
to induce topoisomerase I-mediated DNA cleavage selectively at the
triplex site was analyzed. As summarized in Fig.
10, all TFO conjugates were able
in vitro to selectively direct the action of the
camptothecin derivative at the triplex site and thus induced
sequence-specific DNA cleavage by topoisomerase I. Upon binding of the
TFO-drug conjugate to its specific target, DNA cleavage is strongly
enhanced at the triplex/duplex junction (sites b1 and
b2), where the inhibitor is positioned, and abolished at
other sites (sites a and c-f). The results are
reminiscent of those recently obtained with TFOs conjugated to other
noncamptothecin topoisomerase I poisons such as indolocarbazole and
benzoquinoxaline derivatives (7, 14), but here with the CPT derivatives
both the selectivity and efficacy of cleavage are considerably
reinforced. These findings suggest that the TFO conjugate behaves as a
topoisomerase I poison with a negatively charged tail, which, because
of electrostatic repulsion, eliminates its binding to DNA or
DNA-topoisomerase I sites except at the site where the TFO finds a
target sequence. Binding of the TFO to this DNA site delivers the
topoisomerase I poison selectively to the adjacent cleavage site in
such a position to inhibit the religation of the topoisomerase
I-induced DNA breaks. This strategy offers great promise to enhance the
selectivity of antitumor drugs.
|
The orientation in which the drug is brought in the ternary complex upon triplex formation is an important feature for cleavage stimulation. Collectively, the data demonstrate that the elaboration of a conjugate molecule requires an optimization of the two tethered components but also a precise design of the linker chain to locate the poison in the ternary complex at its preferential site.
Structural data are available for the topoisomerase I-DNA complex (17,
20, 21), but thus far the exact positioning of camptothecin in the
cleavage complex has not yet been determined. Camptothecin does not
interact (or loosely) with either DNA alone or topoisomerase I alone
but does within the cleavage complex (16, 17). Our set of TFO-CPT
conjugates provide a useful molecular tool to probe the structure of
the topoisomerase I-DNA-poison ternary complex. By using TFO and
linkers of variable length and CPT derivatives differently attached to
the TFO, we can change the orientation of the CPT moiety with respect
to the DNA-topoisomerase I complex. The cleavage profile of
TFO16-L6-7CPT resembles the one of TFO18-L6-10CPT with prominent
cleavage at site b1 (Fig. 6), whereas the 18-mer conjugate
of 7CPT (TFO18-L6-7CPT) enhanced cleavage at site b2,
resembling more to the 20-mer 10CPT conjugates (Fig. 5). These two
sites, b1 and b2, are located 4 and 7 bp away from the 3'-end of the triplex formed by the TFO16, respectively. With
both 7CPT and 10CPT, we built a preliminary model for the ternary
complex with the conjugate bound to its triplex site. In the case of
the 7CPT conjugates and the potent TFO16-L4-10CPT, we found that the
distance between the 3'-end of the TFO and the CPT moiety was clearly
too short to enable the insertion of the CPT residue between the 1
and +1 bases of the b1 site. By molecular simulations, Fan
et al. (16) suggested that the camptothecin is
pseudo-intercalated between the bases 1 and +1 in the enzyme-DNA complex. A slightly different configuration has been
proposed by Redinbo et al. (17) on the basis of the crystal
structure of the DNA-topoisomerase I complex. They proposed that the
camptothecin is inserted in the DNA in the space vacated by the base in
+1, a guanine, that flips out of the DNA duplex. We tested both
possibilities, but we were not able to explain our results with an
intercalation model for CPT. There is some degree of flexibility in the
topoisomerase I-DNA complex even at the active site (21), and
therefore, other configurations might be possible. A complex where the
camptothecin moiety is extended in the major groove and points toward
the enzymatic active site could better fit with our findings. On the
other hand, an intercalation model is still possible, if the presence
of the triple helix or the enzyme distorts DNA (21). Different
architectures for the topoisomerase I-DNA-CPT ternary complexes may coexist.
On the basis of gas phase computations, Kerrigan and Pilch (22) have recently proposed a model for CPT interaction with the topoisomerase I-DNA covalent binary complex. Their computations suggest that the A ring of CPT is directed toward the major groove, which would be fully consistent with the data obtained for the TFO conjugate in which the CPT is attached via the 10-position, because any putative structure that leads to site-specific cleavage must involve triplex formation in the major groove. In this context it is interesting to note that the TFO conjugates formed from 7CPT are no less effective in mediating topoisomerase I-dependent cleavage of the target duplex than those involving attachment via the 10-position of CPT, although some adjustments of the length of the tether between the oligonucleotide and CPT moieties are required, as noted above. If the tethered CPTs were associated with the bound topoisomerase I-DNA binary complex via an intercalative interaction, it would be necessary to posit either that the TFO-CPT conjugates attached through positions 7 and 10 had the CPT positioned in the same fashion, necessitating the inclusion of part of the oligomethylene tether within the intercalated complex or else that the nature of the forces that secure the intercalated CPT between DNA base pairs are such that alternative bound orientations may be readily accessible because of small energy differences between them. The latter possibility derives support from two recent publications (8, 9), which demonstrated that the attachment of CPT through position 10 (A ring) to minor groove-binding hairpin pyrrole-imidazole polyamides afforded a conjugate (minor groove binder-CPT) that resulted in topoisomerase I-mediated cleavage at the site of binding of the conjugate. Furthermore, a comparison between TFO-CPT and minor groove binder-CPT conjugates showed that the two class of conjugates are equal both in efficacy and in cleavage sites, despite the fact that the CPT moiety is brought from the major and minor groove side, respectively (8). The possibility that different derivatives of CPT may be capable of binding to the topoisomerase I-DNA covalent binary complex in distinctly different orientations has potentially important implications for the design of novel inhibitors of topoisomerase I function.
Lastly, it is important to emphasize that the TFO-CPT conjugates
described here are extremely potent at inhibiting topoisomerase I. Their action is both sequence-specific and efficient. The positioning of the CPT moiety by triplex formation increases the local drug concentration at the targeted site, and DNA cleavage can be detected using nanomolar concentrations of the conjugate (Figs. 7 and 8). In
terms of concentrations, this is a significant improvement compared
with CPT alone. Moreover, the stability of the covalent topoisomerase
I-DNA cleavage complex is strongly increased (Fig. 9), and this can be
important for the use of topoisomerase I poisons as anticancer agents.
In the future, triple helix-directed targeting of antitumor-active
topoisomerase I poisons may be exploited further to improve the
efficacy of chemotherapeutic cancer treatments by targeting strong and
irreversible topoisomerase I-mediated DNA cleavage selectively at
specific genes.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. J. L. Mergny, J. F. Riou, D. Praseuth, L. Lacroix, and C. Giovannangeli for helpful suggestions.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the European Community and the Ligue Nationale Contre le Cancer (to P. B. A.) and grants from the from the Ligue Nationale Contre le Cancer (Equipe labelisée) (to C. B.) and was supported at the University of Virginia by National Institutes of Health Research Grant CA78415, awarded by the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 33-1-40793859; Fax: 33-1-40793705; E-mail: arimondo@mnhn.fr.
Published, JBC Papers in Press, November 27, 2001, DOI 10.1074/jbc.M110181200
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ABBREVIATIONS |
|---|
The abbreviations used are: CPT, camptothecin; TFO, triplex-forming oligonucleotide; 10CPT, 10-carboxycamptothecin; 7CPT, 7-(2-aminoethyl)camptothecin; DNase I, deoxyribonuclease I.
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REFERENCES |
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RESULTS
DISCUSSION
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