Triple Helix-Forming Oligonucleotides Conjugated to New Inhibitors of

Bioconjugate Chem. 2005, 16, 873−884

873

Triple Helix-Forming Oligonucleotides Conjugated to New Inhibitors of Topoisomerase II: Synthesis and Binding Properties Maria Duca,§ Kahina Oussedik,§ Alexandre Ceccaldi,§ Ludovic Halby,§ Dominique Guianvarc’h,§,† Daniel Dauzonne,‡ Claude Monneret,‡ Jian-Sheng Sun,§ and Paola B. Arimondo§,* Museúm National d’Histoire Naturelle, USM0503 MNHN, UMR5153 CNRS, U565 INSERM, 43 rue Cuvier CP26 75231 Paris Cedex 05 France, and UMR176 CNRS-INSTITUT CURIE, Institut Curie Section de Recherche, 26 rue d’Ulm 75248 Paris Cedex 05 France. Received February 7, 2005; Revised Manuscript Received April 14, 2005

Triplex-forming oligonucleotides (TFOs) are among the most specific DNA ligands and represent an important tool for specific regulation of gene expression. TFOs have also been used to target DNAmodifying molecules to obtain irreversible modifications on a specific site of the genome. A number of molecules have been recognized to target topoisomerase II and stabilize double-stranded cleavage mediated by this enzyme thus determining permanent DNA damage. Among these poisons, etoposide (VP16), a 4′-demethylepipodophyllotoxin derivative, is widely used in cancer chemotherapy. In the aim to design DNA site-specific molecules, three analogues of VP16 (1, 2, and 3), recently described (Duca et al. J. Med. Chem. 2005, 48, 596-603), were attached to TFOs, together with a fourth one, of which the synthesis is reported here. Two different oligonucleotides, differing by the length (a 16mer and a 20-mer), and two different linker arms between the oligonucleotide and the drug were used. The coupling reaction between the drug and the TFO was further improved. For the first time, we also report the synthesis of TFO conjugates bearing two molecules of inhibitor linked to the same oligonucleotide end. In total, 16 new conjugates were synthesized and evaluated for their ability to form triple helices. The loss in triplex stability due to the conjugation of the TFO to compounds that do not interact with DNA is compensated by the presence of the ethylene glycol linker arm. This stabilization effect is more pronounced at the 3′ end than at the 5′ end. All conjugates form a stable triplex selectively on the DNA target at 37 °C and pH 7.2.

INTRODUCTION

Synthetic oligonucleotides can recognize short oligopyrimidine‚oligopurine regions in double-stranded DNA via hydrogen bond formation with purine bases in the major groove, leading to a local triple helix (1-3). Triplexforming oligonucleotides (TFOs1) are among the most specific DNA ligands, together with another class of small molecules represented by synthetic hairpin polyamides that bind to DNA in the minor groove (4). Triplex-based approaches are an attractive mean to achieve targeted gene regulation, gene manipulation, and directed mutagenesis in vitro and in vivo (5-7). Therefore, they represent an important tool for specific regulation of gene expression (1, 2, 6, 8). In fact, TFOs have been described to modulate transcription in vitro and in cell culture for * Corresponding author. Tel: +33 1 40 79 38 59. Fax: +33 1 40 79 37 05. E-mail: [email protected]. § Muse ´ um National d’Histoire Naturelle. ‡ Institut Curie Section de Recherche. † Present address: Laboratoire de Chimie Organique Biologique, Universite´ Paris 6, UMR 7613, 4, place Jussieu, 75005 Paris, France. 1 Abbreviations: TFO, triplex-forming oligonucleotide; 4′DMEP, 4′-demethylepipodophyllotoxin; M, 5′-methyl-2′-deoxycytidine; P, 5′-propynyl-2′-deoxyuridine; VP16, etoposide; bp, base pair; PAGE, polyacrylamide gel electrophoresis; DNase I, 2′-deoxyribonuclease I; EDCI, 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole; L12, 8-[(3aminopropyl)-(7-carboxyheptyl)amino]octanoic acid; L3, ethylene glycol; L18, hexaethylene glycol; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); (PyS)2, dipyridyl disulfide; PPh3, triphenylphosphine.

exogenous and endogenous genes (9-11), to inhibit DNA replication in cells (12), and to direct mutagenesis in vitro and in vivo (13). Furthermore, the sequence-specific recognition properties of TFOs can be exploited to direct DNA cleaving and alkylating agents to chosen DNA sequences (14). In fact, TFOs linked to the appropriate DNA modifying molecule are able to induce a permanent modification in the proximity of the triplex site (7, 15, 16). Interestingly, this strategy has been successfully used to target the action of topoisomerase I inhibitors, such as camptothecin (17-19). Topoisomerases regulate DNA topology in cells and, therefore, are essential for many vital processes such as replication, transcription and chromosome segregation (20, 21). Topoisomerases I and II represent privileged targets for a number of cytotoxic anticancer drugs, which stimulate strand cleavage by topoisomerases at a variety of sites. These drugs are called topoisomerase poisons or inhibitors (22, 23). Whereas topoisomerase I is a monomer and, thus, controls DNA topology by a single-strand breaking of DNA, eukaryotic topoisomerase II is an homodimer and acts by passing an intact DNA double helix through a transient double-stranded break (21). The DNA/topoisomerase II covalent complex, called the cleavage complex, is the key intermediate of the topoisomerase reaction. In this complex, each monomer of the enzyme is covalently linked, through a phosphotyrosine bond, to one strand of the duplex, to the 5′ phosphate of the 4-bp staggered DNA double-stranded break. This intermediate, which has normally a short lifetime, is stabilized by different molecules, such as etoposide or its derivatives,

10.1021/bc050031p CCC: $30.25 © 2005 American Chemical Society Published on Web 06/30/2005

874 Bioconjugate Chem., Vol. 16, No. 4, 2005

Duca et al.

Figure 1. Structure of etoposide and of compounds 1, 2, 3, and 6 used for the coupling to the TFOs.

which are topoisomerase II poisons. These poisons form a stable ternary complex drug/topoisomerase II/DNA and prevent DNA religation. Thereby, they induce breaks in the DNA strands whose accumulation causes cell death (24). So, among the topoisomerase II poisons, some compounds are commonly used as anticancer drugs. Etoposide (VP16, Figure 1), a 4′-demethylepipodophyllotoxin (4′ DMEP) derivative, is one of these drugs and is used as frontline therapy particularly in the treatment of small cell lung cancer, testicular carcinoma, lymphoma and Kaposi’s sarcoma (25, 26). Although VP16 is widely used in chemotherapy, the molecular mechanism of action of this compound and, more generally, of this class of topoisomerase II poisons is not yet fully understood. Moreover, etoposide presents several limitations, such as moderate potency, poor water solubility, development of drug resistance, metabolic inactivation and toxic effects. For these reasons, the structure of VP16 has been extensively modified, thus increasing the information about its structure-activity relationships. In this context, it has been shown that the glucopyranose moiety in the 4-position of etoposide is not essential for activity and that other substituents, such as the aminoalkyl or arylamino chains induce a better antitopoisomerase II activity (27, 28). Recently, we reported that some non glycoside 4′-demethylepipodophyllotoxin derivatives bearing a carbamate chain at the 4 position (4′-demethylepipodophyllotoxin-4-aminoalkyl carbamates, such as 1 and 3, in Figure 1) strongly inhibit topoisomerase II and show antiproliferative effects in vitro and antitumor activity in vivo (29). A new 4′-DMEP derivative, bearing an aminobenzoyl group, has also been synthesized, resulting in an active topoisomerase II poison. To increase the DNA sequence-specificity of these molecules that do not interact with DNA, we covalently attached these derivatives to TFOs, in a way similar to that performed with the topoisomerase I poisons camptothecin and rebeccamycin (19, 30). Sixteen conjugates were synthesized and characterized, using two TFOs (differing by the length) and four VP16 analogues. These derivatives were attached to the 3′ or 5′ end of the TFO

with two linker arms of different length (L18 and L3). Conjugates bearing two molecules of poison at the same end of the TFO were also synthesized and characterized. The stability of the triplex formed by the conjugates was evaluated by gel retardation and DNase I footprinting assays. EXPERIMENTAL PROCEDURES

Methods. 1H NMR and NMR spectra were recorded in chloroform-d or methanol-d4, on a Bruker spectrometer (300 MHz). For all oligonucleotides conjugates, mass determination was accomplished by electrospray ionization on a Q-STAR pulsar I (Appleura) and HPLC purifications were performed upon Agilent 1100 using a Xterra reversed phase C18 column (4.6 × 50 mm, 2.5 µm). Absorbance spectrophotometry was performed on a Uvikon 860 (Kontron). Materials. All chemicals were purchased from Aldrich Chemical Co. All solvents were of analytical grade. 4-(tertButoxycarbonylaminomethyl)benzoic acid was prepared as previously reported (31). Human topoisomerase IIR was purchased from TopoGEN Inc. (Columbus, OH). Oligonucleotides. All oligonucleotides were purchased from Eurogentec (Belgium). Concentrations were determined spectrophotometrically at 25 °C using molar extinction coefficients at 260 nm calculated from a nearest-neighbor model (32). The drug-tethered oligonucleotides were synthesized as described below. The nomenclature of the oligonucleotides and conjugates is as follows. The abbreviation TFO is preceded by a number referring to the length of the oligonucleotide and followed, if attached to the 3′ end, or preceded, if attached through the 5′ end, by the letter L (for linker) and the number of atoms in the linker (L3 ) ethylene glycol; L18 ) hexaethylene glycol; L12 ) 8-[(3-aminopropyl)-(7-carboxyheptyl)amino]octanoic acid), and, finally, by the denomination of the 4′-DMEP derivative. For example, 20TFO-L18-6 stands for the 20-mer TFO linked at its 3′ end through the hexaethylene glycol spacer to compound 6. The orientation of the triple helix is defined as the orientation of the purine-rich strand of the duplex, the

Conjugation of Topoisomerase II Poisons to Oligonucleotides

TFO binding in the major groove in an orientation parallel to the oligopurine strand. Topoisomerase II Poisons. The synthesis of compounds 1, 2, and 3 has been previously described (29). Starting material 4 was obtained as reported (33). Synthesis of 4-β-(4-Boc-aminomethylphenyl)amido-4′-O-demethyl-4-deoxypodophyllotoxin (5). To a solution of 4 (1 g, 2.5 mmol) in anhydrous CH2Cl2 (75 mL), under inert atmosphere, were added 4-(tert-butoxycarbonylaminomethyl)benzoic acid (753 mg, 3 mmol), EDCI (575 mg, 3 mmol) and HOBt (405 mg, 3 mmol). The reaction mixture was further stirred at room temperature for 4 h. The medium was then taken up with CH2Cl2 (25 mL) and water (50 mL). The organic phase was extracted with H2O (3 × 50 mL), then dried (MgSO4) and filtered. This product was further purified by silica gel chromatography using CH2Cl2/methanol 20:1 as the eluent to give pure compound 5 in 70% yield. Rf ) 0.62 (CH2Cl2/methanol 9:1), mp ) 133-135 °C, 1H NMR (CDCl3) ∂: 7.71 (d, 2H, J ) 8.0 Hz, Ph), 7.32 (d, 2H, J ) 7.7 Hz, Ph), 6.80 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.456.32 (m, 1H, 4-NH), 6.30 (s, 2H, 2′,6′-H), 5.97 (d, 2H, J ) 7.1 Hz, CH2O2), 5.50-5.38 (m, 2H, 4-H, 4′-OH), 5.004.88 (m, 1H, NH), 4.58 (d, 1H, J ) 3.2 Hz, 1-H), 4.46 (t, 1H, J ) 8.0 Hz, 11a-H), 4.30 (s, 2H, CH2NH), 3.85 (t, 1H, J ) 9.6 Hz, 11b-H), 3.77 (s, 6H, 3′,5′-OCH3), 3.052.98 (m, 1H, 3-H), 2.95 (dd, 1H, J ) 4.6, 14.3 Hz, 2-H); 1.44 (s, 9H, Boc); MS (CI) m/z: 650 [M + NH4]+. Synthesis of 4-β-(4-Aminomethylphenyl)amido-4′O-demethyl-4-deoxypodophyllotoxin (6). To a solution of 5 (700 mg, 1.1 mmol) in CH2Cl2 (10 mL) was added trifluoroacetic acid (TFA, 855 µL, 11.1 mmol). The reaction mixture was stirred for 5 h, and washed with a cold saturated aqueous NaHCO3 solution then with water until pH 6-7. The organic extract was dried over MgSO4 and concentrated in vacuo at 30 °C. The residue was purified by silica gel column chromatography using a CH2Cl2/methanol 10:1 mixture as the eluent to afford the pure compound 6 in 76% yield. Rf ) 0.17 (CH2Cl2/ methanol 9:1), mp ) 178-180 °C, 1H NMR (CD3OD) ∂: 7.86 (d, 2H, J ) 8.0 Hz, Ph), 7.46 (d, 2H, J ) 7.7 Hz, Ph), 6.82 (s, 1H, 5-H), 6.51 (s, 1H, 8-H), 6.36 (s, 2H, 2′,6′H), 5.93 (d, 2H, J ) 3.5 Hz, CH2O2), 5.52-5.40 (m, 1H, 4-H), 4.58 (d, 1H, J ) 4.8 Hz, 1-H), 4.42 (t, 1H, J ) 8.1 Hz, 11a-H), 3.99 (s, 2H, CH2NH), 3.83 (t, 1H, J ) 9.8 Hz, 11b-H), 3.72 (s, 6H, 3′,5′-OCH3), 3.38-3.22 (m, 1H, 2-H), 3.15-2.98 (m, 1H, 3-H); MS (CI) m/z: 550 [M + NH4]+. Synthesis of the TFO-(4′-DMEP Derivative) Conjugates. The oligonucleotide conjugates were synthesized and purified according to previously reported procedures with little modifications (34). Compounds 1, 2, 3, and 6 have been synthesized with an amino group in order to covalently link them to the phosphorylated poly(ethylene glycol) linker arm at the 3′ end (or the 5′ end) of the oligonucleotides (16TFO and 20TFO). The 3′ or 5′ phosphorylated oligonucleotide (300 µg) was first precipitated as hexadecyltrimethylammonium salt, then dissolved in 50 µL of dry DMSO. Solutions of 4-(N,N-dimethylamino)pyridine (5 mg in 50 µL of DMSO, 41 µmol), dipyridyl disulfide (6.6 mg in 25 µL of DMSO, 30 µmol) and triphenylphosphine (7.9 mg in 25 µL of DMSO, 30 µmol) were added. After 15 min incubation at room temperature, the activated oligonucleotide was rapidly precipitated with 2% LiClO4/acetone and resolubilized in water (50 µL). Triethylamine (3 µL) was added, followed by the drug solution (1 mg in 100 µL of a mixture H2O/DMSO 40:60). After 2 h, the oligonucleotide was precipitated with 2% LiClO4/acetone, rinsed with acetone, then dis-

Bioconjugate Chem., Vol. 16, No. 4, 2005 875

solved in 50 µL of water. Reverse phase HPLC was used to separate the product from the initial oligonucleotide (5 f 40% CH3CN in 0.2 M (NH4)OAc). The product was eluted as a single peak absorbing at 310 nm (oligonucleotide) with retention times between 10 and 12 min. compared to 4-6 min for the initial 16TFO and 20 TFO, respectively. The average yield was 60%. Purity was 100%. 16TFO-L18-1, 1-L18-16TFO MS (ES+) m/z: 5971 [M + H]+ (calculated: 5970), 16TFO-L18-2, 2-L18-16TFO MS (ES+) m/z: 5985 [M + H]+ (calculated: 5984), 16TFOL18-3, 3-L18-16TFO MS (ES+) m/z: 5957 [M + H] (calculated: 5956), 16TFO-L18-6, 6-L18-16TFO MS (ES+) m/z: 6003 [M + H]+ (calculated: 6002), 16TFO-L3-6, MS (ES+) m/z: 5797 [M + H]+ (calculated: 5796), 20TFO-L18-1, 1-L18-20TFO MS (ES+) m/z: 7234 [M + H]+ (calculated: 7233), 20TFO-L18-6, 6-L18-20TFO MS (ES+) m/z: 7266 [M + H]+ (calculated: 7265). Synthesis of the TFO-L18-(6)2 Conjugates. Two molecules of compound 6 were attached to the 3′ phosphate of 16TFO-L18 and 20TFO-L18 using the same procedure as described above but repeating the reaction two times. The products were purified by HPLC (gradient: 0 f 80% CH3CN in 0.2 M (NH4)OAc). The average yield of the reaction was 45% and 100% purity. All conjugates were analyzed by UV spectroscopy, mass spectrometry and gel electrophoresis under denaturing conditions. 16TFO-L18-(6)2 MS (ES+) m/z: 6545 [M + H]+ (calculated: 6544), 20TFO-L18-(6)2 MS (ES+) m/z: 7780 [M + H]+ (calculated: 7779). Synthesis of the 20TFO-L12-(6)2 Conjugates. Two molecules of compound 6 were linked to a trifunctional linker containing two carboxylic groups, the 8-[(3-aminopropyl)-(7-carboxyheptyl)amino]octanoic acid, and then coupled to 3′ phoshophorylated 20TFO. Synthesis of 8-[(3-Boc-aminopropyl)-(7-carboxyheptyl)amino]octanoic Acid (8). 8-Bromooctanoic acid (240 mg, 1.1 mmol) was reacted with N-Boc-1,3-diaminopropane (87 mg, 0.5 mmol, 7) in anhydrous acetonitrile (10 mL) for 48 h at 50 °C in the presence of K2CO3 (210 mg, 1.5 mmol). The solvent was removed under reduced pressure, and the reaction mixture was extracted with methanol/chloroform 9:1 (3 × 5 mL). Organic phases were combined and evaporated in vacuo. The crude product was washed with ether (3 × 5 mL) to afford a white solid in 78% yield. MS (ES+) m/z: 459 [M + H]+ (calculated: 458). Synthesis of L12-(6)2 (9). To a solution of 6 (7 mg, 13 µmol) in anhydrous DMF were added 8 (2.3 mg, 5 µmol), DCC (3.6 mg, 17 µmol) and HOBt 1 M (17 µL, 17 µmol). The reaction mixture was stirred at room temperature for 12 h and then cooled in ether. This led to the formation of a yellow amorphous solid. The crude product was purified by reversed phase HPLC using an acetonitrile gradient (0 f 90% CH3CN with 0.1% of TFA), and obtained in 16% yield. MS (ES+) m/z: 1487 [M + H]+ (calculated: 1486). To a solution of this compound (0.6 mg, 0.4 µmol) in CH2Cl2 (50 µL was added TFA (50 µL). The reaction mixture was stirred at room temperature for 4 h. The mixture was concentrated in vacuo, and the crude product was purified by reversed phase HPLC using an acetonitrile gradient (0 f 90% CH3CN with 0.1% of TFA) to give 9 with a 90% total yield. MS (ES+) m/z: 1387 [M + H]+ (calculated: 1386). Synthesis of 20TFO-L12-(6)2. 3′-Phosphorylated oligonucleotide 20TFO (210 µg, 33 nmol) was first precipitated as hexadecyltrimethylammonium salt, then dissolved in 50 µL of dry DMF. Solutions of 4-(dimethylamino)pyridine (5 mg in 25 µL of DMF, 41 µmol),


dipyridyl disulfide (6.6 mg in 25 µL of DMF, 30 µmol) and triphenylphospine (7.9 mg in 50 µL of DMF, 30 µmol) were added. After 15 min of incubation at room temperature, 9 (0.6 mg, 430 nmol) was added. The mixture was kept at room temperature for 8 h. The oligonucleotide conjugate was then precipitated with 2% LiClO4 in acetone, rinsed with acetone and purified by reversed phase HPLC using a linear acetonitrile gradient (0 f 80% CH3CN in 0.2 M (NH4)OAc). The average yield was of 40%. The oligonucleotide conjugate was characterized by UV spectroscopy, denaturing gel electrophoresis and mass spectrometry. 100% purity was obtained. MS (ES-) m/z: 7776 [M - H]- (calculated: 7776). Gel Retardation Assay. The oligopyrimidine strand of the 29-bp target duplex was 5′ end-labeled with [γ-32P] ATP (Amersham) by T4 polynucleotide kinase (New England Biolabs) according to the manufacturer instructions. Increasing concentrations (from 10 nM to 5 µM) of the triplex-forming oligonucleotides were added to 10 nM of the radiolabeled duplex in 10 mM MgCl2, 50 mM NaCl, 50 mM HEPES pH 7.2, 10% sucrose and 0.5 mg/mL tRNA followed by sample incubation at 37 °C during 2 h. Electrophoresis was performed on a nondenaturing 15% polyacrylamide gel containing 10 mM MgCl2 and 50 mM HEPES pH 7.2 at 37 °C. To quantify the formation of the triplex, the gels were dried and scanned with a Typhoon 9410 (Amersham Biosciences). The concentration of conjugate or TFO necessary to obtain 50% of formed triplex (C50) was calculated, and a mean value corresponding to three to five different experiments is reported. DNase I Protection Assays. The DNA template for DNase I protection assay on the oligopyrimidine-containing strand of the target was obtained by successive digestion of the plasmid described in (18) with Pvu II and Eco RI, yielding a 324-mer fragment suitable for 5′-end labeling by the T4 polynucleotide kinase (Biolabs New England) and γ[32P]-ATP (Amersham) after shrimp alkaline phosphatase treatment, and purified as described. One microliter of DNase I (final concentration 0.03 mg/ mL, Sigma) diluted in 1 mM MgCl2, 1 mM MnCl2, and 20 mM NaCl was added to the radiolabeled duplex (20 nM), preincubated previously for 1 h at 30 °C in 50 mM Tris-HCl, pH 7.2, 20 mM NaCl, 1 mM MgCl2, 2 mM MnCl2, in the absence or in the presence of the conjugates (final reaction volume 10 µL). The reaction was performed for 3 min at 20 °C and stopped by ethanol precipitation. The samples were resuspended in 95% formamide and heated at 95 °C for 4 min before being loaded onto a denaturing 8% polyacryalmide 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 identify DNase I footprinting, the gels were scanned with a Typhoon 9410 (Amersham Biosciences). Topoisomerase II-Mediated DNA Cleavage Assay. Topoisomerase II poisoning by compound 6 was evaluated as previously described (29, 33b). The topoisomerase II poisoning by the conjugates was evaluated as the amount of DNA cleavage by topoisomerase II on a 5′-end radiolabeled 96-bp DNA fragment containing the triplex site. The 96-bp DNA fragment was prepared by 5′-[32P]-end labeling of the EcoRI or Not II/alkaline phosphatase treated plasmid using γ-[32P]-ATP and T4 polynucleotide kinase followed by treatment with NotII/EcoRI, yielding a fragment radiolabeled on the pyrimidine strand (Y) or on the purine strand (R), respectively. The detailed procedure for isolation and purification have been described previously

Duca et al.

(18). Fifty nanomoles of 96-bp radiolabeled DNA fragment was incubated for 1 h at 30 °C in 50 mM Tris-HCl pH 7.0, 120 mM KCl, 1 mM ATP, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, and 30 µg/µL BSA in the presence of the TFO in order to form the triplex or of the drug at 5 µM (total reaction volume 10 µL). Human topo IIR (0.7 units) was added to the duplex preincubated as described above with either the TFO and/or the drugs followed by incubation for 20 min at 30 °C. After ethanol precipitation, all samples were suspended in 6 µL of formamide, heated at 90 °C for 3 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) for 120 min at 65 W. To quantitate the extent of cleavage, the gels were scanned with a Typhoon 9410 (Amersham Biosciences). For the determination of cleavage levels, normalization relative to total loading was performed. Molecular Modeling. Molecular modeling was performed by conformational energy minimization with Jumna program package (version 10.3) (36). The coordinates of DNA triple helices, which were derived from the previously published B-like triple helix, were used for the construction of triplex (37). The published atomic coordinates of yeast topoisomerase II (38) and of gyrase (39) were used to build the model. RESULTS

Oligonucleotides and Topoisomerase II Poisons. The oligonucleotides chosen for this study are a 16-mer (16TFO) and a 20-mer (20TFO) containing 5-methyl-2′deoxycytidine (M) and 5-propynyl-2′-deoxyuridine (P) (Figure 2A). These base modifications were previously shown to enhance triplex formation (40). 16TFO and 20TFO were already shown to form a stable triple helix on a 29-bp target sequence containing a 23-bp oligopyrimidine‚oligopurine sequence at 37 °C, pH 7.2, 50 mM KCl, 10 mM MgCl2 (Figure 2A) (19, 30). The TFOs were covalently attached through three types of linker arms (L3, L12, and L18) to derivatives of 4′-DMEP (Figure 2). Derivatives 1-3 have recently been synthesized and characterized (29). In these compounds the sugar of VP16 is replaced by an aminoalkyl chain linked to the C-4 of 4′-DMEP by a carbamate group (Figure 1). Compounds 1 and 2 bear a 4-N-(3-aminopropyl)carbamate chain or a 4-[N-(3-aminopropyl)-N-methyl]carbamate substituent, respectively. Compound 3 contains a 4-[N-(2-aminoethyl)]carbamate chain and a supplementary modification on the lactone D ring, called retrolactone modification (29, 41). Furthermore, we chose another 4′-DMEP derivative modified on the 4-position (6), containing a secondary aminomethylphenyl side chain with a primary amino group suitable for attachment to the TFO (Figure 1). This compound was designed on the basis of a family of 4′DMEP derivatives, previously described by Zhou et al. (42), bearing aryl chains linked in the 4-position via an amide group. This family of compounds has shown good antitopoisomerase II activity. Compound 6 was synthesized starting from the 4-β-amino-4′-demethylpodophyllotoxin (4), prepared, as previously reported (33), by reaction with 4-(Boc-aminomethyl)benzoic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) (Scheme 1), to provide compound 5, which, after deprotection, leads to 6 in a 76% yield. Compounds 1 and 3 are good topoisomerase II poisons (38% and 48% of inhibition at 20 µM, respectively, expressed as amount of linearized



Figure 2. (A) Sequence of the 29-bp target duplex used for gel retardation assay and sequences of the TFOs (M ) 5′-methyl-2′deoxycytosine, P ) 5-propynyl-2′-deoxyuracile) and chemical structure of the linker arms. The target sequences of 20TFO and 16TFO are surrounded. (B) Chemical structure of synthesized conjugates. The nomenclature of the oligonucleotides and conjugates is as follows: the abbreviation TFO is preceded by a number referring to the length of the oligonucleotide and followed, if attached to the 3′ end, or is preceded, if attached through the 5′ end, by the letter L (for linker) and the number of atoms in the linker, and finally the designation of the 4′-DMEP derivative.

DNA formed upon reaction of supercoiled DNA with human topoisomerase II and compared to 50% at 20 µM

for etoposide used as reference compound), whereas compound 2 is an example of an inactive derivative (8%


Duca et al.

Scheme 1. Synthesis of Compound 6a

a

Reagents: (a) 4-(tert-butoxycarbonylaminomethyl)benzoic acid, EDCI, HOBt, CH2Cl2; (b) TFA, CH2Cl2.

of inhibition at 20 µM) (29). Compound 6 was tested for its activity against topoisomerase II, showing a 25% of amount of linear DNA measured at 20 µM. All these compounds bear a primary amino group at the end of the secondary chain in order to bind them directly to the phosphate group at the extremity of the TFOs. Synthesis of TFO-Topoisomerase II Inhibitors Conjugates. We covalently linked derivatives 1, 2, 3, and 6 to the 3′ or 5′ end of the triple-helix forming oligonucleotides 16TFO and 20TFO. The first generation of conjugates bears one poison per TFO. The chemical structure of these conjugates is depicted in Figure 2B and their synthesis consisted in the activation of the 3′ or 5′ phosphate group of the oligonucleotides in the presence of DMAP, PPh3 and (PyS)2, followed by the addition of the topoisomerase II inhibitors, as previously reported (34). The known procedure used DMSO as solvent for activation of the phosphate group and subsequent reaction with the amino group of the reacting molecule. However, upon use of this protocol, we observed, by HPLC and mass spectrometry, the formation of a secondary product containing both the topoisomerase II poison and a molecule of triphenylphosphine linked to the TFO. To avoid this side-reaction, the TFO activated by DMAP, PPh3, (PyS)2 in DMSO was rapidly precipitated in LiClO4/acetone and the coupling reaction to the topoisomerase II poison was conducted in water. In this manner, triphenylphosphine was removed from the reaction mixture leading exclusively to the desired product with an average yield of 60%. Compounds 1, 2, 3, and 6 were thus conjugated to 16TFO, and 1 and 6 to 20TFO, using hexaethylene glycol (L18) as the linker arm. For the choice of the linker arm between the TFO and the poison, we were aware, from our study of TFO conjugates of topoisomerase I poisons, of the importance of optimizing its length. Therefore, we built a molecular model, based on the crystal structure and the model of the interaction of topoisomerases II with DNA (39, 43). We docked two triple helices symmetrically on both sides of the topoisomerase II to evaluate whether there is steric clash in such a multicomponent complex. This model allowed us to evaluate the distance between the triplex and the catalytic tyrosines in the active site of topoisomerase II. The results suggested that an 18-atom linker would be the best length, and thus a hexaethylene glycol (L18) was used to couple the VP16 derivatives to the TFOs. A shorter linker, ethylene glycol (L3), was also used to conjugate compound 6 to the 3′ end of 16TFO, as a control. In the second generation of conjugates, two VP16 analogues were attached to the same TFO. This is based on the hypothesis of Osheroff’s group (44), concerning the stoichiometry of the complex formed by etoposide and topoisomerase II. In fact, eukaryotic topoisomerase II is constituted by a homodimer and thus bears two active

sites containing two catalytic tyrosines. For this reason, Osheroff proposed that two molecules of etoposide are necessary to generate a double-stranded break and that one molecule of inhibitor leads to a single-stranded break. A conjugate bearing two molecules of topoisomerase II poisons at its end should thus be more efficient than the one bearing just one molecule. Furthermore, the comparison of the two types of conjugates should help to clarify the stoichiometry of the ternary complex formed during the inhibition of the enzyme. Only compound 6 was chosen to apply this strategy, because it gave better yields in the conjugation reaction and, once coupled to the TFO, it resulted more active against topoisomerase II than when compounds 1 and 3 were conjugated (data not shown). Two synthetic pathways were followed (Scheme 2): (1) the introduction of two compounds directly on the same terminal phosphate group of the hexaethylenglycol linker arm (L18) at the 3′ end of the TFO or (2) the attachment of two molecules of 6 to a trifunctional linker arm (L12), containing an amino group for the further coupling to the phosphorylated TFO. In the first pathway, the reaction is conducted as for the synthesis of the mono-conjugates, but it is repeated two times consecutively. We obtained conjugates 16TFO-L18(6)2 and 20TFO-L18-(6)2 with 45% of average yield (Schema 2A). Following the second pathway, we prepared the linker starting from 3-N-Boc-1,3-propanediamine (7) and 8-bromooctanoic acid, which, in the presence of K2CO3 and in acetonitrile, gave the 3-N-Boc-aminopropane-1iminodioctanoic acid (8) (Scheme 2B). Coupling of two molecules of 6 was achieved on the two activated carboxyl groups leading to product 9. This latter, after deprotection of the Boc group by TFA in CH2Cl2, led to 3-aminopropane-1-aminodioctanoic acid (L12) linked through the carboxyl groups to the amino group of 6. Finally, the terminal amino group of 9 was reacted with the activated phosphate of the 20TFO to give 20TFO-L12-(6)2 as final product. Triple Helix Formation by the Conjugates and Topoisomerase II Activity. In the second part of our study the capacity of all synthesized conjugates to form a triple-helix was evaluated. Triplex formation involves major groove binding of the oligonucleotidic part of the conjugates via sequence-specific recognition of the oligopurine strand of the duplex. The binding of the conjugated third strand to the duplex was analyzed by PAGE experiments. Increasing concentration of TFOs were incubated with 10 nM of radiolabeled target duplex in 50 mM HEPES pH 7.2, 10 mM MgCl2, 50 mM NaCl at 37 °C for 2 h (Figure 3). The affinity of all conjugates and of the corresponding TFOs for the target 29 mer duplex was measured and expressed by C50 values, i.e. the concentration of TFO necessary to obtain 50% of triplex. Results are reported in Table 1.



Scheme 2. Synthesis of Conjugates Bearing Two Molecules of 6 at the Same End of the TFO by Methods A and Ba

a Reagents: (a) DMAP, PPh , (PyS) , DMSO; (b) 8-bromooctanoic acid, K CO , CH CN; (c) 6 (2 equiv), DCC, HOBt, DMF; (d) TFA, 3 2 2 3 3 CH2Cl2.

C50 of 16TFO and 20TFO are 0.46 µM and 0.32 µM, respectively. In the case of the 16mer, the introduction of a L18 linker arm at the 3′ end increased the affinity for the duplex, diminishing the C50 4-fold. Interestingly, the shorter linker arm L3 also increased the affinity. The stabilization induced by the linker arm was only 1.4-fold at the 5′ end. In general, the conjugation of the poisons to the 16TFO caused, at both ends, a decrease in the affinity, with the exception of compound 3, which bears the retrolactone modification. Furthermore, conjugation at the 3′ end was more destabilizing than at the 5′ end, even if the 3′ conjugates, however, remained 2 times more stable than the 5′ conjugates. The introduction of two molecules of topoisomerase poison 6 at the 3′ end caused an important loss in triplex affinity (about 40-fold less). The 20 mer TFO, as expected, formed a more stable triple helix. The presence of a linker arm or the conjugation to VP16 analogues had little effect both at the 3′ and 5′ end. The presence of two molecules of 6 at the 3′ end remained, even in this case, very destabilizing with a loss in affinity of 4- and 7-fold for 20TFO-L18-(6)2 and 20TFOL12 -(6)2, respectively. Noteworthy, for all conjugates, 100% of formation of the triple helice was always observed above 5 µM. Evidence for the triplex formation was further confirmed by DNase I footprinting experiments as depicted in Figure 4. The 324-bp duplex fragment radiolabeled at

5′-end of the oligopurine-containing strand was incubated with the TFOs at 5 µM, and subjected to limited DNase I cleavage. DNase I cleavage was strongly inhibited at the target oligopyrimidine‚oligopurine sequence and the footprint was clearly extended with the 20 mer TFOs compared to the 16 mer. As shown in Table 2, all conjugates induce topoisomerase II-mediated DNA cleavage, at the exception of the conjugates of inactive compound 2 that remain inactives. Noteworthy, the experiments were carried out in conditions under which all conjugates form a stable triple helix. TFO conjugates of poison 3 resulted in the less active ones, while the comparison between 16TFOL18-6 and 16TFO-L3-6 confirmed that the hexaethylene glycol (L18) is the best linker arm. Finally, the double conjugates showed a comparable activity as the corresponding mono-conjugates. DISCUSSION

Four 4′-demethylepipodophyllotoxin derivatives were chosen to be covalently attached to triplex-forming oligonucleotides (TFO). As in the case of TFO attached to topoisomerase I poison, these conjugates could be used to target topoisomerase II inhibitors to specific sequences on DNA. Three of them (1, 2, and 3) were recently reported as part of a 4-substituted 4′-DMEP series bearing ami-


Duca et al.

Figure 3. Triplex formation in PAGE experiments. 10 nM of the 29-bp target duplex (RY*, lane 2), 5′-end radiolabeled on the oligopyrimidine-containing strand (Y*, lane 1), was incubated for 2 h at 37 °C in 50 mM HEPES pH 7.2, 10 mM MgCl2 and 50 mM NaCl in the presence of increasing concentrations of (A) 16TFO-L18-3 (0.01, 0.05, 0.1, 0.5, 1, 5 µM lanes 3-8), 3-L18-16TFO (0.01, 0.1, 0.5, 1, 5 µM, lanes 9-14), 1-L18-20TFO (0.01, 0.05, 0.1, 0.5, 1, 5 µM, lanes 15-19); (B) 16TFO-L3-6 (0.05, 0.1, 0.5, 1, 2, 5 µM, lanes 3-8), 20TFO-L18-(6)2 (0.05, 0.1, 0.5, 1, 2, 5 µM, lanes 9-14) and 16TFO-L18-(6)2 (0.05, 0.1, 0.5, 1, 5 µM, lanes 15-19), and then loaded on a 15% nondenaturing polyacrylamide gel. The shifts corresponding to single-stranded DNA (ss), duplex and triplex are indicated by arrows. Table 1. C50 Values for 29R‚29Y* TFO Triplexesa TFOs

C50 (µM)

16TFO 16TFO-L18 16TFO-L18-1 16TFO-L18-2 16TFO-L18-3 16TFO-L18-6 L18-16TFO 1-L18-16TFO 2-L18-16TFO 3-L18-16TFO 6-L18-16TFO 16TFO-L3 16TFO-L3-6 16TFO-L18-(6)2 20TFO 20TFO-L18 20TFO-L18-1 20TFO-L18-6 L18-20TFO 1-L18-20TFO 6-L18-20TFO 20TFO-L18-(6)2 20TFO-L12-(6)2

0.46 ( 0.09 0.11 ( 0.09 0.47 ( 0.10 0.30 ( 0.08 0.17 ( 0.02 0.39 ( 0.08 0.33 ( 0.04 0.74 ( 0.10 0.65 ( 0.05 0.33 ( 0.02 0.59 ( 0.05 0.14 ( 0.02 0.20 ( 0.01 4.1 ( 0.3 0.32 ( 0.02 0.27 ( 0.01 0.38 ( 0.03 0.32 ( 0.03 0.30 ( 0.09 0.27 ( 0.02 0.48 ( 0.08 1.3 ( 0.1 2.2( 0.1

a The C values were calculated as the concentrations at which 50 50% of triplex is formed and an average value corresponding to three different experiments is reported. 29R‚29Y (20 nM) was incubated at 37°C for 2 h with increasing concentrations of TFOs in 50 mM HEPES buffer (pH 7.2) containing 50 mM NaCl and 10 mM MgCl2.

noalkyl chains in the 4-position linked by a carbamate group (29). In this series, some compounds showed an activity on topoisomerase II comparable to etoposide and even better activity in antitumor assays. The most active compounds contained an N,N-dimethyl group at the end of the side chain unsuitable for conjugation to TFOs. We thus designed 1-3 with a NH2 group in the aim to conjugate them directly to the phosphate group at the end of the oligonucleotide. Compounds 1 and 3 are still active as topoisomerase II inhibitors, while compound 2 is an example of inactive compound. Interestingly, when

1 and 3 are conjugated to the TFO, they could eventually become more similar to most active compounds of the series because the terminal NH2 is substituted by the phosphate group of TFO. In addition to these three drugs, we synthesized a new derivative of 4′-DMEP bearing an arylamino side chain linked in the 4-position via an amide group (compound 6). The choice for such a chain is based on a work devoted to a series of derivatives previously synthesized by Zhou et al. (42) that were shown to be active topoisomerase II poisons. All four VP16 analogues were conjugated to a 16mer TFO through a linker arm, a hexaethylene glycol (L18), attached either at the 3′ end or the 5′ end. Analogues 1 and 6 were also linked, via the L18 linker arm, to a 20mer TFO directed against the extended oligopurine‚oligopyrimidine duplex sequence. The previously described coupling reaction (45) was here further improved and carried out in a water/DMSO mixture in order to eliminate all secondary products and to obtain exclusively the desired product. We investigate the influence on the triplex stability of: (i) the length of the TFO, a 16-mer vs a 20-mer, (ii) the linker arm, L3 vs L18, (iii) the modifications of the topoisomerase II poisons (compounds 1, 2, 3, and 6) and (iv) their position on the TFO, attachment to the 3′ end vs 5′ end. Triplex formation by the conjugates was evaluated by gel retardation assay that allowed us to obtain C50 values. The results were further confirmed by DNase I footprinting experiments. Interestingly, the presence of the phosphorylated linker arm alone has a stabilizing effect in the case of 16TFO. The choice for such a long linker chain was based on reported data suggesting that topoisomerase II protected both strands of DNA over 25-bp region, in which the cleavage site was centrally located (46). In addition, Spizner et al. (47) have shown that the enzyme might need an accessible 10-bp double-helical region around the cleavage site to efficiently interact and cleave DNA. According to these observations, a relatively long linker, such as the L18, should be the most appropriate in order



Figure 4. Sequence analysis of the DNase I footprinting experiments on the 324-bp target duplex radiolabeled at the 5′ end of the oligopurine strand in the presence of some TFO conjugates. Control TFOs and conjugates were added at 5 µM. The triplex site and its orientation are indicated by an arrow. Adenine/guanine-specific Maxam-Gilbert chemical cleavage reactions were used as markers.

to access the drug to the catalytic site of topoisomerase II and to prevent steric hindrance between the triplehelical structure and the enzyme. Noteworthy, the tethered 4′ DMEP derivatives are now able to bind to DNA through the oligonucleotidic part of the conjugate and specifically to the triplex target (as shown by the DNase footprinting experiments). The tethering to the TFOs does not hamper the activity of the 4′ DMEP derivatives against topoisomerase II (Table 2). In the case of compound 2, an example of inactive topoisomerase II poison, it remains inactive even when conjugated to the TFO. This is different from what is observed with camptothecin, a topoisomerase I poison, for which the conjugation of an inactive derivative to the TFO is able to convert it to an active topoisomerase I poison (48). Concerning triplex formation, the conjugation of the poison decreased, as expected, the affinity of the TFOs for the target duplex, the worst cases being the conjugates bearing two molecules of poisons linked to the same TFO end. This destabilization effect is more pronounced at the 3′ end than at the 5′ end, even if 3′ conjugates remain more stable than the respective 5′ conjugates. An exception is compound 3, bearing the retrolactone, which was comparable in affinity to the

16TFO attached to the linker arm both in the 3′ end or 5′ end. The observed destabilization effect can be explained by the mechanism of action of these poisons. In fact, these molecules do not interact with DNA (29), but rather with the enzyme (49). The absence of interaction of these latter conjugates with DNA can explain, in part, the absence of stabilization of the triple-helical structure. Other topoisomerase II poisons families, such as amsacrines and antracyclines that intercalate into DNA, induce a stabilization of the triple-helical structure when conjugated to a TFO. In this context, it was shown that daunomycin strongly stabilized triplex formation when conjugated to the 5′ end (10, 50); although no effect was observed at the 3′ end of the triplex (10). In the case of amsacrine (mAMSA), an acridine derivative inhibitor of topoisomerase II, a slight stabilization of the triplex structure was obtained when an amsacrine derivative was tethered to the 5′ end of the TFO (51). Linkage of the amsacrine derivative to the 3′-end of the TFO decreased triplex stability and the destabilizing effect was less pronounced with the conjugate containing a longer linker side chain (an hexaethylene glycol). Interestingly, in this study, the 5′ end conjugates were 2-fold less stable than the 3′ end conjugates. This can be


Duca et al.

Table 2. Normalized Percentage (%) of Total DNA Cleavage on a Radiolabeled 96-bp DNA Fragment by Topoisomerase II in the Presence of the Free Drugs or Conjugated to the TFOsa drug/TFOb

normalized percentage, %

1 2 3 6 16TFO-L18-1 16TFO-L18-2 16TFO-L18-3 16TFO-L18-6 1-L18-16TFO 2-L18-16TFO 3-L18-16TFO 6-L18-16TFO 16TFO-L3-6 16TFO-L18-(6)2 20TFO-L18-1 20TFO-L18-6 1-L18-20TFO 6-L18-20TFO 20TFO-L18-(6)2 20TFO-L12-(6)2

42 8 40 45 27 5 20 30 26 3 15 25 24 27 30 32 23 22 30 26

a

An average value corresponding to three different experiments is reported. b Drugs were evaluated at 50 µM, conjugates at 1 µM at the exception of the double conjugates that were evaluated at 10 µM.

explained, in part, by the fact that triplex formation seems to nucleate at the 5′ end of the triplex (52, 53), and thus the presence of the drug at this end might interfere with the nucleation step and be more destabilizing. In agreement, the above-cited studies, together with previous ones (54), have demonstrated that the stabilizing effect of an intercalating drug is more important at the 5′ triplex/duplex junction than on the 3′ side. Finally, in the case of the more stable 20TFO, very little differences were observed with the exception of the double conjugation that was very destabilizing for triplex formation, even if the effect was less than in the case of 16TFO. The higher affinity of the 20mer compared to the 16mer seems to abolish the differences due to the energetic penalty for the presence of the DNA-noninteracting molecule. At last, we synthesized TFO conjugates containing two molecules at the same end, either both attached directly to the same phosphate at the end of the linker arm of the TFO (TFO-L18-(6)2), or each one attached to a linker arm further conjugated at the same phosphate at the extremity of the TFO (20TFO-L12-(6)2). These are the first examples of such a type of conjugates. All three double conjugates showed an important loss in triplex stability, but 100% of formation of triplex was still observed at pH 7.2 and 37 °C at high concentrations (>5 µM). Moreover, the importance of such conjugates resides in the fact that topoisomerase contains two subunits and thus two active sites (two Tyr804 for human topoisomerase II). This implies that there may be two sites available for the drug in every cleavage complex (44). These double-poison conjugates should represent a priori an important tool for the investigation of such a molecular mechanism. We observed, however, that their ability to stimulate DNAcleavage on a 96-bp radiolabeled DNA fragment is comparable to that of the mono-conjugate (Table 2). In conclusion, we synthesized a new poison of topoisomerase II derived from 4′-DMEP. This compound, together with other 4′-DMEP analogues previously reported, was conjugated to triplex-forming oligonucleotides with an average yield of 60%. We then evaluated the

stability of the formed triplex and defined the best features for stability. Unexpectedly, the phosphorylated poly(ethylene glycol) linker arm increased the triplex stability and compensated for the presence of the 4′ DMEP derivatives that do not interact with DNA. The 20-mer conjugates form triplexes with comparable stability as the unlinked 20TFO. In the case of the 16mers, conjugation to the 3′ end is preferable to conjugation to the 5′ end. Finally, the conjugates retain the antitopoisomerase II activity. ACKNOWLEDGMENT

This work was supported by grants from Ligue Nationale contre le Cancer and Ministe`re de la Recherche et de l′Industrie (ACI “Molećules et Cibles The´rapeutiques”) (to P.B.A.), fellowships from Foundation de France (to D.G.), Association pour la Recherche contre le Cancer (ARC) (to M.D.), and Assocation pour la Recherche sur les Tumeurs de la Prostate (ARTP) (to K.O.). LITERATURE CITED (1) Faria, M., and Giovannangeli, C. (2001) Triplex-forming molecules: from concepts to applications. J. Gene Med. 3, 299-310. (2) Praseuth, D., Guieysse, A. L., and He´le`ne, C. (1999) Triple helix formation and the antigene strategy for sequencespecific control of gene expression. Biochim. Biophys. Acta 1489, 181-206. (3) Giovannangeli, C., and He´le`ne, C. (2000) Triplex-forming molecules for modulation of DNA information processing. Curr. Opin. Mol. Ther. 2, 288-96. (4) Dervan, P. B., and Edelson, B. S. (2003) Recognition of the DNA minor groove by pyrrole-imidazole polyamides. Curr. Opin. Struct. Biol. 13, 284-99. (5) Faria, M., Wood, C. D., White, M. R., He´le`ne, C., and Giovannangeli, C. (2001) Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells. J. Mol. Biol. 306, 15-24. (6) Vasquez, K. M., Narayanan, L., and Glazer, P. M. (2000) Specific mutations induced by triplex-forming oligonucleotides in mice. Science 290, 530-3. (7) Guieysse, A. L., Praseuth, D., Giovannangeli, C., Asseline, U., and He´le`ne, C. (2000) Psoralen adducts induced by triplexforming oligonucleotides are refractory to repair in HeLa cells. J. Mol. Biol. 296, 373-83. (8) Giovannangeli, C., and He´le`ne, C. (2000) Triplex technology takes off. Nat. Biotechnol. 18, 1245-6. (9) Besch, R., Giovannangeli, C., and Degitz, K. (2004) Triplexforming oligonucleotidesssequence-specific DNA ligands as tools for gene inhibition and for modulation of DNA-associated functions. Curr. Drug Targets 5, 691-703. (10) Carbone, G. M., McGuffie, E., Napoli, S., Flanagan, C. E., Dembech, C., Negri, U., Arcamone, F., Capobianco, M. L., and Catapano, C. V. (2004) DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene. Nucleic Acids. Res. 32, 2396-410. (11) Carbone, G. M., Napoli, S., Valentini, A., Cavalli, F., Watson, D. K., and Catapano, C. V. (2004) Triplex DNAmediated downregulation of Ets2 expression results in growth inhibition and apoptosis in human prostate cancer cells. Nucleic Acids. Res. 32, 4358-67. (12) Diviacco, S., Rapozzi, V., Xodo, L., He´le`ne, C., Quadrifoglio, F., and Giovannangeli, C. (2001) Site-directed inhibition of DNA replication by triple helix formation. FASEB J. 15, 2660-8. (13) Seidman, M. M., and Glazer, P. M. (2003) The potential for gene repair via triple helix formation. J. Clin. Invest. 112, 487-94. (14) Arimondo, P. B., Boutorine, A., and Franç ois, J. C. (2002) Oligonucleotide-conjugates targeted to single-stranded and double-stranded nucleic acids. Recent Res. Dev.. Bioconjugate Chem. 1, 29-53.

Conjugation of Topoisomerase II Poisons to Oligonucleotides (15) Guillonneau, F., Guieysse, A. L., Nocentini, S., Giovannangeli, C., and Praseuth, D. (2004) Psoralen interstrand cross-link repair is specifically altered by an adjacent triplestranded structure. Nucleic Acids. Res. 32, 1143-53. (16) Grigoriev, M., Praseuth, D., Guieysse, A. L., Robin, P., Thuong, N. T., He´le`ne, C., and Harel-Bellan, A. (1993) Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. Proc. Natl. Acad. Sci. U.S.A. 90, 3501-5. (17) Arimondo, P. B., Bailly, C., Boutorine, A. S., Ryabinin, V. A., Syniakov, A. N., Sun, J. S., Garestier, T., and He´le`ne, 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. (18) Arimondo, P. B., Bailly, C., Boutorine, A. S., Moreau, P., Prudhomme, M., Sun, J. S., Garestier, T., and He´le`ne, C. (2001) Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site. Bioconjugate Chem. 12, 501-9. (19) Arimondo, P. B., Boutorine, A., Baldeyrou, B., Bailly, C., Kuwahara, M., Hecht, S. M., Sun, J. S., Garestier, T., and He´le`ne, C. (2002) Design and optimization of camptothecin conjugates of triple helix-forming oligonucleotides for sequencespecific DNA cleavage by topoisomerase I. J. Biol. Chem. 277, 3132-40. (20) Wang, J. C. (1996) DNA topoisomerases. Annu. Rev. Biochem. 65, 635-92. (21) Champoux, J. J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369-413. (22) Burden, D. A., and Osheroff, N. (1998) Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400, 139-54. (23) Capranico, G., and Binaschi, M. (1998) DNA sequence selectivity of topoisomerases and topoisomerase poisons. Biochim. Biophys. Acta 1400, 185-94. (24) Wilstermann, A. M., and Osheroff, N. (2003) Stabilization of eukaryotic topoisomerase II-DNA cleavage complexes. Curr. Top Med. Chem. 3, 321-38. (25) Hande, K. R. (1998) Etoposide: four decades of development of a topoisomerase II inhibitor. Eur. J. Cancer 34, 151421. (26) Hande, K. R. (2003) Topoisomerase II inhibitors. Cancer Chemother Biol. Response Modif. 21, 103-25. (27) Terada, T., Fujimoto, K., Nomura, M., Yamashita, J., Wierzba, K., Yamazaki, R., Shibata, J., Sugimoto, Y., Yamada, Y., Kobunai, T., and et al. (1993) Antitumor agents. 3. Synthesis and biological activity of 4 beta-alkyl derivatives containing hydroxy, amino, and amido groups of 4′-Odemethyl-4-desoxypodophyllotoxin as antitumor agents. J. Med. Chem. 36, 1689-99. (28) Zhu, X. K., Guan, J., Tachibana, Y., Bastow, K. F., Cho, S. J., Cheng, H. H., Cheng, Y. C., Gurwith, M., and Lee, K. H. (1999) Antitumor agents. 194. Synthesis and biological evaluations of 4-beta-mono-, -di-, and -trisubstituted aniline-4′-Odemethyl-podophyllotoxin and related compounds with improved pharmacological profiles. J. Med. Chem. 42, 2441-6. (29) Duca, M., Guianvarc’h, D., Meresse, P., Bertounesque, E., Dauzonne, D., Kraus-Berthier, L., Thirot, S., Leónce, S., Pierre´, A., Pfeiffer, B., Renard, P., Arimondo, P. B., and Monneret, C. (2005) Synthesis and biological study of a new series of 4′-demethylepipodophyllotoxin derivatives. J. Med. Chem. 48, 593-603. (30) Arimondo, P. B., Moreau, P., Boutorine, A., Bailly, C., Prudhomme, M., Sun, J. S., Garestier, T., and He´le`ne, C. (2000) Recognition and cleavage of DNA by rebeccamycin- or benzopyridoquinoxaline conjugated of triple helix-forming oligonucleotides. Bioorg. Med. Chem. 8, 777-84. (31) Gangjee, A., Devraj, R., McGuire, J. J., and Kisliuk, R. L. (1995) Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines. J. Med. Chem. 38, 3798-805. (32) Cantor, C. R., Warshaw, M. M., and Shapiro, H. (1970) Oligonucleotides interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers 9, 1059-1077.

Bioconjugate Chem., Vol. 16, No. 4, 2005 883 (33) (a) Lee, K. H. J. Med. Chem. 1991, 34, 3346. (b) Guianvarc’h, D., Duca, M., Boukarim, C., Kraus-Berthier, L., Leonce, S., Pierre, A., Pfeiffer, B., Renard, P., Arimondo, P. B., Monneret, C., and Dauzonne, D. (2004) Synthesis and biological activity of sulfonamide derivatives of epipodophyllotoxin. J. Med. Chem. 47, 2365-74. (34) Grimm, G. N., Boutorine, A. S., and He´le`ne, C. (2000) Rapid routes of synthesis of oligonucleotide conjugates from nonprotected oligonucleotides and ligands possessing different nucleophilic or electrophilic functional groups. Nucleosides Nucleotides Nucleic Acids. 19, 1943-65. (35) Arimondo, P., Boukarim, C., Bailly, C., Dauzonne, D., and Monneret, C. (2000) Design of two etoposide-amsacrine conjugates: topoisomerase II and tubuline polymerization inhibition and relation to cytotoxicity. Anticancer Drug Des. 15, 413-21. (36) Lavery, R., and Sklenar, H. (1988) The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63-91. (37) Ouali, M., Letellier, R., Adnet, F., Liquier, J., Sun, J. S., Lavery, R., and Taillandier, E. (1993) A possible family of B-like triple helix structures: comparison with the Arnott A-like triple helix. Biochemistry 32, 2098-103. (38) Berger, J. M., Gamblin, S. J., Harrison, S. C., and Wang, J. C. (1996) Structure and mechanism of DNA topoisomerase II. Nature 379, 225-32. (39) Morais Cabral, J. H., Jackson, A. P., Smith, C. V., Shikotra, N., Maxwell, A., and Liddington, R. C. (1997) Crystal structure of the breakage-reunion domain of DNA gyrase. Nature 388, 903-6. (40) Froehler, B. C., Jones, R. J., Cao, X. D., and Terhorst, T. J. (1993) Oligonucleotide derived from 5-(1propynyl)-2-O-allyluridine and 5-(1-propynyl)-2-O-allyl-cytidine: Synthesis and RNA duplex formation. Tetrahedron Lett. 34, 1003-1006. (41) Meresse, P., Magiatis, P., Bertounesque, E., and Monneret, C. (2003) Synthesis and antiproliferative activity of retroetoposide. Bioorg. Med. Chem. Lett. 13, 4107-9. (42) Zhou, X. M., Wang, Z. Q., Chen, H. X., Cheng, Y. C., and Lee, K. H. (1993) Antitumor agents. 125. New 4 betabenzoylamino derivatives of 4′-O-demethyl-4-desoxypodophyllotoxin and 4 beta-benzoyl derivatives of 4′-O-demethylpodophyllotoxin as potent inhibitors of human DNA topoisomerase II. Pharm. Res. 10, 214-9. (43) Fass, D., Bogden, C. E., and Berger, J. M. (1999) Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands. Nat. Struct. Biol. 6, 322-6. (44) Bromberg, K. D., Burgin, A. B., and Osheroff, N. (2003) A two-drug model for etoposide action against human topoisomerase IIalpha. J. Biol. Chem. 278, 7406-12. (45) Grimm, G. N., Boutorine, A. S., Lincoln, P., and He´le`ne, C. (2001) Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation. Nucleosides Nucleotides Nucleic Acids. 20, 909-14. (46) Lee, M. P.; Sander, M.; Hsieh, T. (1989) Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites. J. Biol. Chem. 264, 21779-87. (47) Spitzner, J. R., Chung, I. K., and Muller, M. T. (1995) Determination of 5′ and 3′ DNA triplex interference boundaries reveals the core DNA binding sequence for topoisomerase II. J. Biol. Chem. 270, 5932-43. (48) 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 HelixForming Oligonucleotides. Biochemistry 44, 4171-4180. (49) Kingma, P. S., Burden, D. A., and Osheroff, N. (1999) Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance. Biochemistry 38, 3457-61. (50) Garbesi, A., Bonazzi, S., Zanella, S., Capobianco, M. L., Giannini, G., and Arcamone, F. (1997) Synthesis and binding

884 Bioconjugate Chem., Vol. 16, No. 4, 2005 properties of conjugates between oligodeoxynucleotides and daunorubicin derivatives. Nucleic Acids. Res. 25, 2121-8. (51) Arimondo, P., Bailly, C., Boutorine, A., Asseline, U., Sun, J. S., Garestier, T., and He´le`ne, C. (2000) Linkage of a triple helix-forming oligonucleotide to amsacrine-4-carboxamide derivatives modulates the sequence-selectivity of topoisomerase II-mediated DNA cleavage. Nucleosides Nucleotides Nucleic Acids. 19, 1205-18. (52) Alberti, P., Arimondo, P. B., Mergny, J. L., Garestier, T., He´le`ne, C., and Sun, J. S. (2002) A directional nucleationzipping mechanism for triple helix formation. Nucleic Acids. Res. 30, 5407-15.

Duca et al. (53) Arimondo, P. B., Barcelo, F., Sun, J. S., Maurizot, J. C., Garestier, T., and He´le`ne, C. (1998) Triple helix formation by (G,A)-containing oligonucleotides: asymmetric sequence effect. Biochemistry 37, 16627-35. (54) Sun, J. S., Francois, J. C., Montenay-Garestier, T., SaisonBehmoaras, T., Roig, V., Thuong, N. T., and He´le`ne, 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, 9198-202.

BC050031P

Triple Helix-Forming Oligonucleotides Conjugated to New Inhibitors of

Recommend Documents