Triple Helix-Forming Oligonucleotides Conjugated to Indolocarbazole

Triple Helix-Forming Oligonucleotides Conjugated to Indolocarbazole Poisons Direct Topoisomerase I-Mediated DNA Cleavage to a Specific Site...
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Bioconjugate Chem. 2001, 12, 501−509

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Triple Helix-Forming Oligonucleotides Conjugated to Indolocarbazole Poisons Direct Topoisomerase I-Mediated DNA Cleavage to a Specific Site Paola B. Arimondo,† Christian Bailly,*,† Alexandre S. Boutorine,‡ Pascale Moreau,§ Michelle Prudhomme,§ Jian-Sheng Sun,‡ The´re`se Garestier,‡ and Claude He´le`ne‡ INSERM U524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place Verdun, 59045 Lille, France; Laboratoire de Biophysique, UMR 8646 CNRS-Muse´um National d′Histoire Naturelle, INSERM U201, 43 rue Cuvier, 75231 Paris cedex 05, France; and SEESIB, UMR 6504 CNRS, Universite´ Blaise Pascal, 63177 Aubie`re, France . Received May 21, 2001

Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazoleoligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 µM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.

INTRODUCTION

Sequence-specific modulation of gene expression can be achieved by oligonucleotides that bind to double-helical DNA via triplex formation (1-5). These triple-helical DNA structures offer new prospects to modulate the interactions between proteins and DNA. Cleaving or cross-linking agents have been tethered to triple helixforming oligonucleotides [TFOs] in order to target chemical reactions to specific sequences on DNA (1, 2, 6-12). An attractive option for biological applications would be the recruitment of a cellular enzyme whose action on DNA would be directed by triplex formation to perform a sequence-specific DNA cleavage. One such enzyme is topoisomerase I, which is an ubiquitous nuclear enzyme involved in the control of DNA topology (13). The reaction of topoisomerase I with double-stranded DNA produces a covalent 3′-phosphorotyrosyl adduct, usually referred to as the cleavage complex. Under physiological conditions, the covalent intermediate is barely detectable, because a fast religation step occurs after relaxation of DNA constraints. A number of drugs, such as the antitumor alkaloid camptothecin [CPT], can convert topoisomerase I into a cell poison by blocking the * Corresponding author: Christian Bailly, INSERM U524, IRCL, Place Verdun, 59045 Lille, France, phone: +33-320169218; fax: +33-3-20169229; e-mail: [email protected]. † IRCL. ‡ CNRS-Muse ´ um National d′Histoire Naturelle. § Universite ´ Blaise Pascal.

religation step, thereby enhancing the formation of persistent DNA breaks responsible for cell death (13, 14). Apart from the camptothecins, several classes of antineoplastic drugs have been shown to stabilize DNAtopoisomerase I complexes. One of the most promising series of antitumor agents targeting topoisomerase I is represented by indolocarbazole analogues of the antibiotic rebeccamycin [RBC], which strongly stimulate topoisomerase I-mediated DNA cleavage (15-17). In contrast to the camptothecins, RBC and analogues intercalate in DNA even in the absence of topoisomerase I. Structureactivity relationship studies have provided key information concerning the stabilization of DNA-topoisomerase I covalent complexes by indolocarbazoles (15, 18-21). These considerations prompted us to attach a rebeccamycin derivative to a TFO in order to recruit topoisomerase I and direct its cleavage to specific DNA sequences. Upon intercalation into DNA, the attached indolocarbazole chromophore can stabilize the triplehelical structure and, in addition, can direct the topoisomerase I enzymatic reaction to particular sites at proximity to the triple helix binding site (22, 23). In a previous study, Matteucci and co-workers (24) have shown that a triplex-forming oligonucleotide conjugated to camptothecin is able to direct topoisomerase I-mediated DNA cleavage to a specific site. In this study, three indolocarbazole derivatives, R6, R0, and R95 (see Figure 1 for chemical structures) were covalently attached to the 3′-end of a 16-nt triplex-forming oligonucleotide and their potencies for topoisomerase I recruitment compared. The

10.1021/bc000162k CCC: $20.00 © 2001 American Chemical Society Published on Web 06/15/2001

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Figure 1. (A) Sequence of the TFOs and the 77-bp target used in this study. The TFO binds in the major groove to the oligopurine strand of the duplex and parallel to it. The target site is in bold and underlined. The 77-bp duplex target sequence was inserted between the BamH I and EcoR I sites of pBSK((). M ) 5-methyl-2′-deoxycytidine, P ) 5-propynyl-2′-deoxyuridine, rU ) uridine, L6 ) hexaethylene glycol, L3 ) triethylene glycol, L2 ) diethylene glycol. (B) Structure of the indolocarbazole-TFO derivatives used in this study. U ) uracil. The TFO-R6/TFO-R0 was synthesized with the spacer linked at one end to the 3′-phosphate of the TFO and at the other end to a uridylic acid. The glycol function of the ribose was oxidized by NaIO4 and reacted with the NH2 group linked to the maleimide function of R6 or R0. For the synthesis of the TFO-R95 conjugate, the hexaethylene glycol linker at the 3′-end of the TFO was phosphorylated and after activation by N-methylimidazole, dipyridyl disulfide, and triphenylphosphine, it was reacted with the NH2-terminal group of the 1-aminopropyl side chain of the B cycle of R95 (see reference (22) for details).

compounds differ by the nature of their functional groups, which allowed us to use different types of linkage to tether them to the TFO.1 These different conjugates were used to further investigate the parameters involved in the interaction between topoisomerase I and the triplehelical structure in order to rationally design topoisomerase I poison-oligonucleotide conjugates that efficiently cleave DNA at specific sites. 1 Abbreviations: TFO ) triplex-forming oligonucleotide, bp ) base pair, nt ) nucleotide, ‚ ) Watson-Crick base pairing, x ) Hoogsteen or reverse Hoogsteen base pairing, ssDNA ) singlestranded DNA, dsDNA ) double-stranded DNA, PAGE ) polyacrylamide gel electrophoresis, RBC ) rebeccamycin, CPT ) camptothecin, DNase I ) deoxyribonuclease I, Kd ) dissociation constant defined as: Kd ) ([Duplex][Oligonucleotide])/ [Triplex], M ) 5-methyl-2′-deoxycytidine, P ) 5-propynyl-2′deoxyuridine, rU ) uridine, L2 ) diethylene glycol, L3 ) triethylene glycol, L6 ) hexaethylene glycol.

EXPERIMENTAL PROCEDURES

Drugs, Oligonucleotides, and DNA Fragment. The synthesis of the indolocarbazoles R-6, R-0, and R-95 have been previously described (18, 22, 25). 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. They were attached to the 3′-end of the TFO as described in Figure 1. Oligonucleotides were purchased from Eurogentec and purified using quick spin columns and sephadex G-25 fine (Boehringer, Mannheim). Concentrations were determined spectrophotometrically at 25 °C using molar extinction coefficients at 260 nm calculated from a nearest-neighbor model (26). The rebeccamycin derivatives-oligonucleotide conjugates were synthesized and purified as reported in (22) and their structure is shown in Figure 1. The NH2 group

Triplex-Directed DNA Cleavage by Topoisomerase I

of the aliphatic chain of R-95 was covalently linked to the phosphorylated hexaethylene glycol linker arm at the 3′ end of TFO (Figure 1B) [22]. The 3′ phosphorylated oligonucleotide (150 µg) was first precipitated as a hexadecyltrimethylammonium salt and the oligonucleotide salt was then dissolved in 50 µL of dry DMSO. A 5 µL sample of N-methylimidazole and 25 µL each of dipyridyl disulfide and triphenylphosphine solutions [1.2 M DMSO] were added. After 15 min incubation at room temperature, 5 µL of triethylamine was added followed by the drug solution [20 µL, 30 mM in DMSO]. After 20 min, the oligonucleotide was precipitated with LiClO4. Reverse phase HPLC using a linear acetonitrile gradient (5-80% CH3CN in 0.2 M (NH4)OAc) was used to separate the product from the initial oligonucleotide. A Lichrosorb C-18 column (4.6 × 25 cm) and Shimadzu CR4-A Chromopack instrument were used (average yield 60%). The product was eluted as a single peak absorbing both at 310 nm (oligonucleotide) and 410 nm (rebeccamycin) with a retention time of 11.4 min compared to 10.4 min for initial TFO. To covalently link R6 or R0 to the 3′-end of the oligonucleotide, we used the 16-mer TFO bearing at the 3′end an uridine linked through an poly(ethylene glycol) linker arm [22]. 300 nmoles of oligonucleotide (ethanol precipitated), and 6.5 mg of sodium periodate (NaIO4) were dissolved in 100 µL of 200 mM sodium acetate, pH 5.2. After 20 min incubation at room temperature and in the dark, 100 µL of 2 M potassium chloride was added in order to precipitate the excess of periodate. The pellet was washed several times with 50 µL of H2O. All water phases were collected, and the oligonucleotide was precipitated with ethanol. It was then was dissolved in 50 µL of 200 mM NaOAc, pH 5.2 and added to 1 mg of the indolocarbazole compound R6 or R0 dissolved in 50 µL of DMF. The solution was incubated for 12 h at 4 °C in the dark. After ethanol precipitation, the oligonucleotide was dissolved in water and treated with 0.5 mg of sodium borohydrate and again precipitated with ethanol. The conjugates were recovered by reverse phase HPLC with a Lichrosorb C-18 column (4.6 × 25 cm) using a linear acetonitrile gradient (5-40% CH3CN in 0.2 M (NH4)OAc) and a Shimadzu CR4-A Chromopack instrument (average yield 40%). The product was eluted as a single peak absorbing both at 310 nm (oligonucleotide) and 410 nm (rebeccamycin). Retention times: TFO-L2-R6, 13.1 min vs 11.1 min; TFO-L3-R6, 13.0 min vs 11.2 min; TFOL6-R6, 13.6 min min. vs 11.7 min.; TFO-L6-R0, 13.0 min vs 11.7 min for the drug-free oligonucleotide. All conjugates were analyzed by UV spectroscopy and gel electrophoresis under denaturing conditions. In each case, the ratio of A310 nm/A410 nm from the UV-vis absorbance spectrum corresponded to a 1:1 oligonucleotide: rebeccamycin complex. On a 20% denaturing gel electrophoresis only one band migrating slower than the one of the initial TFO was observed. The band revealed blue fluorescence as the free rebeccamycin derivative. The plasmid pBSK(() was bought from Promega, and the 77-bp target duplex was inserted between the Bam HI and Eco RI sites. The digestion of the plasmid by PvuII and Eco RI yielded a 324-mer fragment suitable for 3′-end labeling by the Klenow polymerase and R[32P]ddATP (Amersham). The detailed procedures for isolation, purification and labeling of this duplex DNA fragment have been described previously (27). 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

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MgCl2, 0.5 mM DTT, 0.1 mM EDTA, and 30 µg of BSA, in the presence of the TFO (at the indicated concentration) in order to form the triplex (total reaction volume 20 µL). To analyze the topoisomerase I DNA cleavage products, 10 units of enzyme (Life Sciences) were added to the duplex, preincubated as described above with either the TFO or/and the drugs, followed by incubation for 30 min at 30 °C. DNA-topoisomerase I cleavage complexes were dissociated by addition of SDS (final concentration 0.25%) and of proteinase K (Sigma) to 250 µg/mL, followed by incubation for 30 min at 55 °C. After ethanol precipitation, all 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 1xTBE 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, a normalization relative to total loading was performed. RESULTS AND DISCUSSION

Prior to investigating the effects of triplex formation with oligonucleotides conjugated to topoisomerase I poisons on DNA cleavage by topoisomerase I, the ability of the conjugates to form the triple helix was determined. Triple Helix Formation by TFO-RBC Conjugates. As previously described (22), modified 16-nt TFO, containing 5-propynyl-2′-deoxyuracils [P] and 5-methyl2′-deoxycytosines [M] (Figure 1), can form a stable triple helix on the target sequence (Figure 1, bold and underlined), at 37 °C and at pH 7.2 in a buffer containing 50 mM KCl, 10 mM MgCl2. We covalently attached the 3′end of the oligonucleotide to the indolocarbazole derivatives R6, R0, and R95 as described in Figure 1B. Triplex formation involves major groove binding of the conjugates via recognition of the oligopurine strand of the duplex (28). The binding should allow the planar indolocarbazole ring to intercalate at the duplex/triplex junction as previously described for other intercalating agents (29, 30). Initially the RBC derivatives were attached to the TFO by a hexaethylene glycol linker (L6). The choice for such a long linker chain was based on previously reported data suggesting that topoisomerase I protected both strands of DNA over a 15-19-bp region in which the cleavage site was centrally located (31). This suggested that the enzyme might need an accessible 7-9-bp double helical region around the cleavage site, and therefore we reasoned that a relatively long linker (L6) should be used to allow the drug to get access to the catalytic site of topoisomerase I and to prevent steric hindrance between the TFO and the enzyme. But as shown below, this linker can be further shortened to improve topoisomerase I-mediated cleavage on the basis of the crystal structure of topoisomerase I. The indolocarbazole drugs, R6, R0, and R95, bear different functional groups used to tether the indolocarbazole chromophore to the TFO. R0 and its glycosylated analogue R6 were attached through reaction of the NH2 group on the nitrogen of the maleimide function with the ribose of an uridine attached at the 3′end of the oligonucleotide through a linker arm (Figure 1). In contrast, the R95 derivative was linked to a phosphate attached to the hexaethyleneglycol linked to the 3′-end of TFO via the NH2 group of the 1-aminopropyl side chain attached to the B cycle of the drug.

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Figure 2. (A) Sequence analysis of the topoisomerase I-mediated cleavage products on the 324-bp target duplex (50 nM) 3′-end radiolabeled on the oligopyrimidine-containing strand. Adenine/guanine-specific Maxam-Gilbert chemical cleavage reactions were used as markers. The positions of the cleavage sites are indicated (sites a to j). Lane 1, target duplex; duplex incubated with topoisomerase I (10 units) in the absence (lane 2) or in the presence of 5 µM camptothecin (lane 3), 5 µM R95 (lane 4), 5 µM R6 (lane 5), 5 µM TFO (lane 6), 5 µM TFO and R6 (lane 7), 5 µM TFO-L6-R6 (lane 8), and 5 µM TFO-R95 (lane 9). The triple helix region and the nucleotide positions along the radiolabeled oligopyrimidine strand are indicated. (B) The ratio between the intensity of cleavage in the presence of TFO-L6-R6 and the intensity in the presence of the untethered R6 is represented on a logarithmic scale at different sites along the DNA fragment used as a substrate for topoisomerase I cleavage (data from lanes 8 and 5 of Figure 2A).

The binding of the conjugated third strands to the duplex was analyzed by gel retardation experiments, and the affinity of the TFOs for the duplex was measured. The following dissociation constants (Kd) were determined (as described in (22)): TFO: 5.3 ( 0.5 µM; TFO-R6: 0.20 ( 0.06 µM; TFO-R0: 0.50 ( 0.1 µM; TFO-R95: 0.85 ( 0.07 µM. Only the RBC-conjugates were studied, because as intercalating agents the RBC derivatives could greatly modify the stability of the triplex. Indeed, all conjugates bind more strongly to the duplex than the corresponding drug-free oligonucleotide. These results showed that the TFO-drug conjugates formed stable triple helices in the absence of topoisomerase I. Of the three conjugates, only TFO-R6 and TFO-R0 are comparable, because they share the same type of attachment point to the TFO. A 2-fold higher binding affinity was observed with the R6 conjugate, presumably due to the presence of the β-glucose residue attached to the indole nitrogen on ring B, which has been previously shown to stabilize drug-DNA complexes in the case of the free drugs (19). Topoisomerase I-Mediated DNA Cleavage in the Presence of the Triple Helix. The 3′-end-radiolabeled restriction fragment (324-bp) bearing the 16-bp triple helix target site was incubated in the presence of TFOs and topoisomerase I. After protease treatment, DNA

cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Figure 2 shows the results obtained when using the duplex target labeled at the 3′end of the oligopyrimidine-containing strand. We compared the different topoisomerase I poisons, R6, R95, and R0, and their TFO conjugates. Lane 2 shows the cleavage pattern of topoisomerase I alone in the absence of TFO and topoisomerase I poisons. Two sites are present near the 3′ triplex/duplex junction (sites b) and one near the 5′ junction (site a). There are also several other sites at some distance from the triplex binding site. The indolocarbazoles R95 (lane 4) and R0 (data not shown) are weak topoisomerase I poisons and enhance only slightly topoisomerase I-mediated cleavage (15, 19). In the presence of R6 (lane 5), sites b1 and b2, located at 4-bp and 7-bp from the 3′ triplex end, respectively, are strongly enhanced, like that of site a on the 5′-side of the triplex at 8-bp from the 5′ triplex end. Additional sites were also detected in the oligopyrimidine‚ oligopurine region and in the regions marked c, d, e, f, g, h, i, and j in Figure 2. Sequence analysis showed that most of these cleavage sites correspond to TVG sites, in agreement with previous findings showing that RBC derivatives stimulate DNA cleavage by topoisomerase I

Triplex-Directed DNA Cleavage by Topoisomerase I

preferentially at sites having a T and a G on the 3′-and 5′-sides of the cleaved bond, respectively (15). The topoisomerase I cleavage profile changed only slightly in the presence of the triplex formed with the untethered oligonucleotide TFO (lane 6 compared to lane 2). The formation of the triple helix protected the duplex against enzymatic cleavage in this region. Site a at 8-bp from the 5′ duplex/triplex junction and sites b at the 3′triplex/duplex junction were still present. The addition of R6 to the triplex (lane 7) restored cleavage at sites a and b. Again there were no cleavage sites in the triple helix region. It is interesting to observe that the presence of the triplex enhanced the cleavage at the site situated at 4-bp from the 3′ triplex end (site b1) compared to cleavage at the b2 site. This could be due to an increase of local drug concentration at this site as a consequence of its intercalation at the triplex/duplex junction. The very strong site characteristic of R6 at 33-bp from the 3′-end of the triplex was still present (region c), as well as the other sites d to j. Triplex formation with the TFO-L6-R6 conjugate (lane 8) diminished the cleavage at sites located far away from the triple helix (c, d, e, f, g, h, i, k) and at site a on the 5′-side of the TFO. Only site b1, at the 3′-end of the triple helix, was maintained and enhanced. This is consistent with a sequence-specific recruitment of topoisomerase I by triplex formation with TFO conjugates bearing topoisomerase I poisons at the 3′ end. The TFO bearing the drug R6 at the 3′-end favors DNA cleavage by the enzyme only on the 3′-side of the triplex and decreases the other characteristic sites of the drug: this is clearly due to the targeting of the drug by triplex formation specifically to the b1 site at the 3′-end of the triple helix. In fact, the triple helix formed by the uncoupled oligonucleotide stimulates cleavage equally at the three sites adjacent to the triplex (lane 6, sites a, b1, and b2) and upon addition of the free drug, all the characteristic cleavage sites of the drug are present (lane 7, sites a, b, c, d, e, f, g, h, i, and j). In Figure 2B is shown the ratio of the intensities measured with the conjugate TFO-L6-R6 and the free drug (R6) at 5 µM. Only site b1 shows an enhanced cleavage whereas all other sites reveal a decrease in cleavage efficiency, demonstrating that the drug-TFO conjugate is able to target the topoisomerase I poison specifically near the triplex site and to increase the efficiency of cleavage of the agent at this site. The enhancement observed at site b1 was 3-fold for the TFOL6-R6 as compared to free R6. It was only 2.6-fold when compared to the TFO+R6 mixture at 5 µM concentration. The decrease at site b2 was 5- and 2-fold, respectively. The TFO-R95 conjugate was much less potent at inhibiting topoisomerase I than its congener TFO-L6-R6 (compare lanes 9 and 8, Figure 2A). Nevertheless, cleavage at the expected site b1 remained visible and enhanced. The cleavage at site a on the 5′ end of the triple helix and at site b2 located 7-bp away from the 3′ end of the triplex was less efficient compared to the free R95 (lane 4). Again this is consistent with a triple helixinduced effect. As for the TFO-R95 conjugate, in the presence of the triplex formed by the TFO-R0 conjugate, cleavage was maintaned only at site b1 (data not shown). Therefore, TFOs bearing topoisomerase I poisons at the 3′-end favor DNA cleavage by the enzyme only on the 3′-side of the triplex and decreases the other characteristic sites of the free drug. This is clearly due to the targeting of the poison by triplex formation specifically to site b1 at 4-bp from the 3′-end of the triple helix. The lower efficacy of TFO-R95 and TFO-R0 compared to TFO-R6 is entirely consistent with the data presented

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Figure 3. Molecular model for the complex formed between the TFO-L3-R6 conjugate and the topoisomerase I bound to DNA. The atomic coordinates of the topoisomerase I/DNA complex published by Rebinbo et al. (32) were used to construct this model. The R-helix, β-sheets, and the backbone are colored in red, yellow, and brown, respectively. The duplex region of the DNA is represented in white; the double helix site involved in triple helix formation is represented in pink (oligopurine strand) and in light blue (oligopyrimidine strand). The third strand moiety of the TFO-L3-R6 conjugate is in yellow. The extended linker chain (L3) positions the drug moiety at 4-bp from the triplex/duplex junction with the indolocarbazole chromophore intercalated in the double helix at the cleavage site. The glucose moiety of the drug is located in the minor groove, in a suitable position to interact with the Arg364 residue of topoisomerase I. Closer view of the model showing the drug binding pocket around the topoisomerase cleavage site.

above indicating that R95 and R0 are much less efficient than R6 both in terms of triplex stabilization and stimulation of topoisomerase I cleavage. As mentioned above, TFO-R95 not only lacks the essential glucose residue but the F-ring on the indolocarbazole chromophore is oriented toward the minor groove, which is an unfavorable orientation for optimal topoisomerase I inhibition. The F ring is presumed to interfere with the DNA-enzyme complex from the major groove (19). This might explain why the cleavage efficiency of the R95 conjugate is poor. The cleavage profile obtained with R0 was similar to that found with R95. The absence of the sugar moiety is presumably the most important element

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Figure 4. Sequence analysis of topoisomerase I-mediated cleavage products with R6 conjugates containing linker arms of different length. Adenine/guanine-specific Maxam-Gilbert chemical cleavage reactions were used as markers. The positions of the cleavage sites are indicated (sites a to j). Lane 1, target duplex; duplex incubated with topoisomerase I (10 units) in the absence (lane 2) or in the presence of 5 µΜ TFO (lane 3), 5 µΜ TFO-L2-R6 (lane 4), 5 µΜ TFO-L3-R6 (lane 5), 5 µΜ TFO-L6-R6 (lane 6), 5 µΜ R6 (lane 7), and 5 µΜ TFO and 5 µΜ R6 (lane 8). The triple helix region and the nucleotide positions along the radiolabeled oligopyrimidine strand are indicated. The band with an asterisk corresponds to a labeling artifact and was used as loading control.

responsible for the weak effect on topoisomerase I because the results obtained with the conjugate TFOR0 were comparable to those obtained with TFO-R95. This corroborates the findings that the sugar moiety is important for both binding to DNA and poisoning of topoisomerase I (15, 19). At this stage, the results suggest that topoisomerase I recruitment I by a TFO coupled to a potent poison can be achieved with RBC derivatives, with efficiencies corresponding to the potency of the free drug. Furthermore, we have shown that the attachment point of the poison to the TFO orients the drug in the drug/topoisomerase I/DNA complex and thus changes the intensity of DNA cleavage. The conjugates could thus be used to study the orientation of the drug in the ternary complex, by measuring the efficiency of cleavage as a function of the attachment point of the drug to the TFO. Design of an Optimized Linker for Oligonucleotide-Rebeccamycin Conjugates. The reported crystal structure of the complex formed between a 22-bp DNA duplex and a large fragment of human topoisomerase I (32) allowed us to build a molecular model where the drug moiety of the TFO-R6 conjugate was positioned in the catalytic site of the enzyme. According to this model, a triplex could be formed without steric clash with topoisomerase I at the 3′-side of the cleavage site, while the 5′-side of the cleavage site was not accessible (Figure 3). A pyrimidine motif triple helix was docked into the crystal structure at 4-bp from the cleavage site, called b1, identified in Figure 2A. The indolocarbazole derivative was attached through the linker arm to the 3′-end of the TFO and placed in the cleavage site. The model in Figure 3 shows the intercalated indolocarbazole derivative with its methoxy-glucose moiety attached to the B-ring of the chromophore. The sugar lies in the minor groove, in a suitable position to interact with amino acid Arg364, and to a lesser extent with the Asp533 residue of topoisomerase I. This model was used to investigate further the influence of the linker chain between the TFO and the drug. The modeling suggested that a triethylene glycol linker (L3) would be more appropriate than the hexaethylene glycol linker (L6) to position the drug at the b1 cleavage site on the 3′ side of the triplex. Moreover, this shorter linker should permit major groove binding of the TFO 4-bp away from the cleavage site without steric interference with topoisomerase I.

To test this prediction, we synthesized the conjugates TFO-L3-R6 and the analogue TFO-L2-R6 bearing a diethylene glycol linker (Figure 1B). Both conjugates were tested in parallel with the TFO-L6-R6 conjugate initially constructed. The 324-bp restriction fragment bearing the 16-bp triple helix target site was incubated in the presence of the conjugates and topoisomerase I. After protease treatment, DNA cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis (Figure 4A). Lane 2 shows the cleavage pattern of topoisomerase I alone in the absence of TFO and drugs. As above-described, the presence of the triple helix formed by the untethered oligonucleotide (lane 3) has only a slight effect on the topoisomerase I cleavage pattern. In the presence of the R6 compound alone (lane 7), the two sites b1 and b2 were detected on the 3′ side of the triplex site, as well as site a on the opposite side of the triplex. Additional sites were observed in the regions marked c to j. The addition of the TFO (lane 8) did not affect the cleavage profile seen with the free drug. The TFO-[L2, L3, L6]-R6 conjugates (lanes 4-6) enhanced cleavage specifically at site b1. Cleavage at sites a at the 5′ end of the triple helix and b2 located 7-bp away from the 3′ end of the triplex are strongly reduced compared to the free R6 compound (lane 7), as the other sites c to j. Again this is consistent with a triple helix-induced effect. In the R6 series, the 9 atoms linker (L3) produced a 4- and 2.6-fold stronger topoisomerase I-mediated DNA cleavage than TFO-L2-R6 and TFO-L6-R6, respectively, and 8-fold stronger than R6 alone. The TFO-R6 conjugate with the shorter linker arm enhanced cleavage at site b1 about 8 times as compared to free R6 and 3 times as compared to TFO+R6 at 5 µM concentration. The experimental data fully agrees with the modeling predictions: the triethylene glycol is the most appropriate linker to allow positioning of the R6 compound at the same site in the complex. Collectively, the data demonstrate that the elaboration of a conjugate molecule requires an optimization of the two linked components but also a precise design of the linker chain. Concentration Effect. Next we studied the efficacy of the TFO-drug conjugate compared to that of the free drug. We compared topoisomerase I-mediated DNA cleavage in the presence of increasing concentrations of R6 (from 0.01 µM to 5 µM, Figure 5A, lanes 5 to 10) and of

Triplex-Directed DNA Cleavage by Topoisomerase I

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Figure 5. Analysis of the topoisomerase I-mediated cleavage products as a concentration function. (A) Sequence analysis of the topoisomerase I-mediated cleavage products. Duplex (lane 1) incubated with topoisomerase I (lane 2) in the presence of 5 µM TFO (lane 3), of 5 µM TFO and R6 (lane 4), or of increasing concentration of R6 (0.01; 0.05; 0.1; 0.5; 1 and 5 µM, lanes 5 to 10) or of TFO-L3-R6 (0.01; 0.05; 0.1; 0.5 and 1 µM, lanes 11 to 15). Other details as in Figure 2. (B) Plot of the cleavage intensities of R6 (circles) and TFO-L3-R6 (triangles) at sites h, c, b1, and a (from Figure 5) versus the conjugate concentration.

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TFO-L3-R6 (from 0.01 µM to 1 µM, lanes 11 to 15). The conjugate induces efficient cleavage even at a concentration as low as 0.01 µM (lane 11) compared to R6 alone (lane 5). Figure 5B compares the percentage of cleavage in the presence of R6 (red circles) and in the presence of TFO-L3-R6 (blue triangles) at sites h, c, b1, and a as a function of the concentration. The R6 conjugate not only strongly enhanced topoisomerase I cleavage at the triplex site b1 but it is 10-fold more efficient at 10 nM than the poison alone at the same concentration. In contrast, at sites other than b1 cleavage was abolished. CONCLUSION

The designed TFO-indolocarbazole conjugates are able to direct the action of the topoisomerase I poison to a specific site in vitro. They induce sequence-specific DNA cleavage by topoisomerase I. Upon binding of the TFOdrug conjugate to its specific target, DNA cleavage is strongly enhanced at the triplex/duplex junction where the poison is positioned and strongly decreased at other sites. Three novel aspects were studied. First, the combined computational and experimental approach helped to identify the determinants, predominantly drug inhibitory potency and length of the linker arm, which shape the TFO-directed DNA cleavage mediated by topoisomerase I. Second, the comparison between the R0 and R6 conjugates provides important information on the role of the sugar residue attached to the indolocarbazole chromophore. Third, the positioning of the drug by triplex formation increases its local concentration at this site, and thus the conjugates induce cleavage in the nanomolar concentration range as compared to micromolar for the free drug. In the future, triple helix-directed targeting of antitumor-active topoisomerase I poisons, such as camptothecin and indolocarbazole analogues, may be exploited further to improve the efficacy of chemotherapeutic cancer treatments by reducing drug toxicity and targeting topoisomerase I-induced cleavage to appropriately chosen genes. In addition to their utilization as therapeutic tools, TFO conjugates could be used as research tools: these conjugates provide a new approach for understanding the interactions involved in the topoisomerase I/drug/DNA ternary complex. In fact, with this approach, by varying the attachment sites and the functional groups as well as the nature and length of the linker, the drug can be located in different and controlled orientations with respect to topoisomerase and DNA. ACKNOWLEDGMENT

The authors thank Dr. J. L. Mergny, J.F. Riou, D. Praseuth, L. Lacroix, and C. Giovannangeli for helpful suggestions. This work was supported by grants (to P.B.A.) from the European Community and the Ligue Nationale Contre le Cancer; (to C.B.) from the from the Ligue Nationale Contre le Cancer (Comite´ du Nord). LITERATURE CITED (1) Le Doan, T., Perrouault, L., Praseuth, D., Habhoub, N., Decout, J.-L., Thuong, N. T., Lhomme, J., and He´le`ne, C. (1987) Sequence specific recognition, photo-cross-linking and cleavage of the DNA double helix by an oligo R thymidylate covalently linked to an azidoproflavine derivative. Nucl. Acids Res. 15, 7749-7760. (2) Moser, H. E., and Dervan, P. B. (1987) Sequence specific cleavage of double helical DNA by triple helix formation. Science 238, 645-650. (3) Maher, L. J., III. (1996) Prospects for the therapeutic use of antigene oligonucleotides. Cancer Invest. 14, 66-82.

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