**2.1.2 Bis-intercalators**

Bisacridine A (BisA) (Figure 1) is a DNA unwinding bis-intercalator deriving from acridine orange by cyclization of two acridine planar chromophores using polyammonium bridges. Initially designed to interact with ss- rather than ds-DNA (Teulade-Fichou et al., 1995), BisA shifts duplexes DNA toward hairpins and destabilizes dsDNA (Slama-Schwok et al., 1997).

Fig. 1. DNA intercalators that destabilize the DNA helix: structures and 3D orientation of morpholino-doxorubicin (Top, [mmdbId:52942]) or ellipticine (Bottom, [mmdbId:52189]).

#### **2.2 DNA alkylators as helix destabilizing agents**

98 Selected Topics in DNA Repair

Stability of DNA double helix is mainly due to reversible non-covalent hydrogen bonds between Watson-Crick base-pairs. Local or global denaturation (melting or breathing) of the double-stranded DNA (dsDNA) helix is dispensable for different cellular processes: DNA replication, transcription and repair (Choi et al., 2004; Schneider et al., 2001). DNA melting is affected by sequence (AT- or GC-rich portions, some successive base pairs arrangements) and their specific tilt, roll, twist effects (Benham, 1996; Dornberger et al., 1999; Krueger et al., 2006), the formation of local hairpins, 3D structures at terminal regions of the DNA helix (Putnam et al., 1981) or internal portions of B- to Z-DNA transition (Harvey, 1983). Such locally opened sites are good substrates for, or are generated by, some cellular proteins: DNA helicases (Betterton & Julicher, 2005), single strand binding proteins (SSBP) such as replication protein A (RPA) (Wold, 1997), UP1 and myeloma helix-destabilizing protein (Herrick & Alberts, 1976; Planck & Wilson, 1980), GAPDH-related protein P8 (Karpel & Burchard, 1981), High Mobility Group (HMG) proteins (Butler et al., 1985), c-Abl kinase (David-Cordonnier et al., 1998, 1999), HIV-1 nucleocapsid protein (Narayanan et al., 2006),

UHRF-1 protein (Arita et al., 2008). Besides large DNA opening, small modifications such as base flipping locally perturb DNA stability (Hornby & Ford, 1998) during mismatches or repair proteins interaction from NER (Cao et al., 2004), BER (Bellamy et al., 2007; Tubbs et

Besides naturally occurring DNA breathing, unzipping is induced by clinically used or potential anti-tumor compounds. The vast majority of DNA-interacting compounds stabilize the DNA double helix; only a very few of them displays the pecular ability to destabilize DNA helix. In this latter group, most belong to DNA intercalating or alkylating

Historically, the first DNA intercalating compound evidencing DNA destabilization properties was acridine orange (Figure 1), a well-known dsDNA intercalating compound and a strong single-stranded DNA (ssDNA) binder. It emitted green fluorescence emission from dsDNA binding and red luminescence from ssDNA interaction. Acridine orange enhances the global helix stability but exerts local denaturation of DNA (Kapuscinski & Darzynkiewicz, 1983; 1984; Darzynkiewicz et al, 1983). Ellipticine and adriamycin (Figure 1) also induce local unzipping of the DNA and bind ssDNA (Zunino et al., 1972), in contrast with ethidium bromide (BET), highly specific to dsDNA and stabilizing DNA. Intercalation of acridine orange, ellipticine and adriamycin progressively unzip the DNA helix preferentially in heterochromatin, ribosomes and polysomes (Darzynkiewicz et al., 1983).

Bisacridine A (BisA) (Figure 1) is a DNA unwinding bis-intercalator deriving from acridine orange by cyclization of two acridine planar chromophores using polyammonium bridges. Initially designed to interact with ss- rather than ds-DNA (Teulade-Fichou et al., 1995), BisA shifts duplexes DNA toward hairpins and destabilizes dsDNA (Slama-Schwok et al.,

B transcription factor (Mura & McCammon, 2008) and

al., 2007) or DNA methylases/demethylases (Sundheim et al., 2008).

**2.1 DNA intercalators as helix destabilizing agents** 

**2. DNA destabilizing compounds** 

prion protein (Bera et al., 2007), NF-

families.

**2.1.1 Mono-intercalators** 

**2.1.2 Bis-intercalators** 

1997).

Some DNA alkylating drugs could also locally destabilize DNA double helix. Some of those are used/developed as anticancer drugs such as cisplatin and metal-derivatives, or more recently the benzoacronycine derivative S23906-1. They contrast with most DNA alkylating agents used or not in chemotherapy that stabilize DNA helix (for instance mitomycin C, dinuclear platinum, nitrogen mustards or ecteinascidine 743) (Basu et al., 1993; David-Cordonnier et al., 2005; Fridman et al., 2003; Kasparkova et al., 1999). Electrophilic alkylating drugs react at nucleophilic positions of G-C or A-T bp with preferential targets: N7 position of dG or dA and O6 position of dG in the major groove, N3 positions of dG or dA and exocyclic NH2 group on C2 of dG (also called N2) in the minor groove (Figure 2).

Fig. 2. Position of the reactive sites of some DNA alkylators on G-C or A-T base pairs.

DNA Helix Destabilization by Alkylating Agents: From Covalent Bonding to DNA Repair 101

and binding kinetics (C.X. Zhang & Lippard, 2003). From this series, Ru-CYM presents the highest DNA helix destabilization activity, together with the smaller unwinding angle in supercoiled plasmid DNA (7° *vs*. 14° for Ru-BIP, Ru-DHA and Ru-THA), in correlation with its lack of intercalation and the formation of monoadducts at N7-dG (Nováková et al., 2009). New Ru-derivatives monodentate-Ru(II) and [Ru(terpy)(4,4'-(COLysCONH2)2bpy)Cl]3+ also destabilize DNA (Nováková et al., 2010; Triantafillidi et al., 2011). For gallium-complexed compounds, interaction of trivalent Ga-cations with calf-thymus DNA resulted in major helix destabilization with perturbations at A-T base pairs sites (R. Ahmad et al. 1996).

Fig. 3. Structure of cisplatin and other transition-metal agents as DNA destabilizing drugs

DNA interaction of carcinogen, adduct formation and their repair processes are widely studied using carcinogens from environmental and tobacco smoke. Some of them have the ability to destabilize the DNA helix: BPDE ((+/-)-*anti-*benzo[*a*]pyrene-7,8-dihydrodiol-9,10-

The smoke carcinogen benzo[*a*]pyrene (BaP) is metabolized into several enantiomers of BPDE that covalently bond the exocyclic NH2 group of guanines to form a bulky adduct in the minor groove of the DNA helix, resulting in its destabilization (Zou & Van Houten, 1999). Due to the orientation of the reactive epoxide group on asymmetric carbons, several enantiomers are produced. The most carcinogenic is 10S(+)-*trans*-*anti*-BPDE N2-dG adduct followed by the stereo-isomeric 10R(+)-*cis*-*anti*-BPDE-N2-dG adducts. Covalent bonding to DNA is associated with base-displaced intercalation where the bulky adduct prevents the hydrogen bonding of the amino group of guanine with the opposite cytosine. This results in

and 3D orientation [mmdbId:47796] (cisplatin) and [mmdbId:69361] (oxaliplatin).

**2.2.2 Carcinogens as DNA destabilizing agents** 

epoxide) and 4-OHEN (4-hydroxyequilenin-O-quinone) (Figure 4).

#### **2.2.1 Cisplatin and other transition-metal antitumor agents as DNA destabilizing drugs**

Fortuitously discovered in 1965, cisplatin (or *cis*-diaminedichloridoplatinum(II) is used in clinic since 1978 and is still frequently administrated in combinatory chemotherapies as one of the most effective anticancer drugs against solid tumors (Figure 3). Cisplatin forms interand intra-strand crosslinks as well as monovalent adducts. Those lesions occur primarily though covalent bonding to the N7 atom of guanines. The most common lesions are intrastrand crosslink at the 5'-GG (65%) or 5'-AG (25%) dinucleotides and inter-strand crosslinks (5-8%). This latter lesion is more frequent using transplatin (12%), *trans-*PtCl2(NH3)(quinoline) and *trans-*PtCl2(NH3)(thiazole) derivatives (up to 30%) (Figure 3). Cisplatin-induced intra-strand crosslinks at GpG base-pairs result in a strong DNA helix bending toward the major groove with an angle of 55-78° associated with DNA distortion, resulting in a destabilization of the Watson-Crick base pairing and local denaturation of the DNA helix (bending at 45° and unwinding by 79+/-4°) (Bellon, 1991; Malinge et al., 1994; Todd & Lippard, 2010). In platinated-GpG intra-strand crosslinks, the distortion varies and depends on the sequence context, with up to a 7 bp distortion for 1,3-intrastrand crosslinks within a TGTGT sequence (Kasparkova et al., 2008a). Such destabilization was found to be enthalpic, but not entropic, in origin. Similarly, when occurring at 5'-TGGT site, cisplatin adducts decrease the melting temperature of the DNA by more than 10°C which is much higher than that induced on 5'-CGGT and 5'-AGGC sequences (~6°C) (Malina et al., 2007).

Such effects are not observed with transplatin which does not change the transition entropy or enthalpy and, consequently, does not destabilize the DNA helix (Kasparkova et al., 2008a). Third-generation platinum antitumor derivative oxaliplatin (Figure 3) induces greater DNA bending, unwinding and helix destabilization than cisplatin, whereas JM118 (Figure 3) induces DNA destabilization profiles similar to that of cisplatin (Kostrhunova et al., 2010). JM118 is the major metabolite of satraplatin (JM216), the first orally administered platinum drug that also evidenced promising therapeutic activities in prostate cancer. JM118 induces a DNA bending with an angle of 28° toward the major groove, an angle smaller than that obtained with cisplatin for the same sequence (34°) (Kostrhunova et al., 2010).

Besides the nature of the platinated drug, the surrounding DNA sequence is also of major importance for helix stability. Indeed, monofunctional platinum adducts at 5'-TGC triplet induces major DNA destabilization (Brabec et al., 1992) but none at 5'-AGT or 5'-TGA triplet (Schwartz et al., 1989). DNA is not the unique nucleic acid destabilized by platinated derivatives as evidenced using *cis*-[PtCl(NH3)2(OH2)]+, *cis*-[PtCl(NH3)(c-C6H11NH2)(OH2)]+ and *trans*-[PtCl(NH3)(quinoline)(OH2)]+ (Figure 3) which not only destabilize ds-DNA but also ds-RNA (Tm of -11°C and -5°C, respectively) (Hägerlöf et al., 2006).

Besides platinum derivatives, ruthenium compounds were developed as anti-cancer drugs. NAMI-A was the first ruthenium derivative that entered phase I clinical trials in 1999 as an anti-metastatic drug (Bergamo et al., 2002), followed by KP1019 (FFC14A) in 2003 (Hartinger et al., 2008). Two gallium compounds, gallium maltolate and KP46 (FFC11), also entered phase I clinical trials in 2003 (Lum et al., 2003). As for cisplatin, ruthenium derivatives evidenced DNA destabilization properties. This is particularly well described for Ru-CYM ([(6--cymene)Ru(II)(en)-(Cl)]+ and Ru-BIP, Ru-DHA or Ru-THA as biphenyl, dihydroanthracene or tetrahydroanthracene derivatives, respectively (Figure 3). Such organometallic ruthenium(II) arene complexes were rationally designed for chemotherapy with the idea that changing platinum for ruthenium would provide additional coordination sites in the octahedral complexes to modify the oxidation rate and change ligand affinity

Fortuitously discovered in 1965, cisplatin (or *cis*-diaminedichloridoplatinum(II) is used in clinic since 1978 and is still frequently administrated in combinatory chemotherapies as one of the most effective anticancer drugs against solid tumors (Figure 3). Cisplatin forms interand intra-strand crosslinks as well as monovalent adducts. Those lesions occur primarily though covalent bonding to the N7 atom of guanines. The most common lesions are intrastrand crosslink at the 5'-GG (65%) or 5'-AG (25%) dinucleotides and inter-strand crosslinks (5-8%). This latter lesion is more frequent using transplatin (12%), *trans-*PtCl2(NH3)(quinoline) and *trans-*PtCl2(NH3)(thiazole) derivatives (up to 30%) (Figure 3). Cisplatin-induced intra-strand crosslinks at GpG base-pairs result in a strong DNA helix bending toward the major groove with an angle of 55-78° associated with DNA distortion, resulting in a destabilization of the Watson-Crick base pairing and local denaturation of the DNA helix (bending at 45° and unwinding by 79+/-4°) (Bellon, 1991; Malinge et al., 1994; Todd & Lippard, 2010). In platinated-GpG intra-strand crosslinks, the distortion varies and depends on the sequence context, with up to a 7 bp distortion for 1,3-intrastrand crosslinks within a TGTGT sequence (Kasparkova et al., 2008a). Such destabilization was found to be enthalpic, but not entropic, in origin. Similarly, when occurring at 5'-TGGT site, cisplatin adducts decrease the melting temperature of the DNA by more than 10°C which is much higher than that induced on 5'-CGGT and 5'-AGGC sequences (~6°C) (Malina et al., 2007). Such effects are not observed with transplatin which does not change the transition entropy or enthalpy and, consequently, does not destabilize the DNA helix (Kasparkova et al., 2008a). Third-generation platinum antitumor derivative oxaliplatin (Figure 3) induces greater DNA bending, unwinding and helix destabilization than cisplatin, whereas JM118 (Figure 3) induces DNA destabilization profiles similar to that of cisplatin (Kostrhunova et al., 2010). JM118 is the major metabolite of satraplatin (JM216), the first orally administered platinum drug that also evidenced promising therapeutic activities in prostate cancer. JM118 induces a DNA bending with an angle of 28° toward the major groove, an angle smaller than that obtained with cisplatin for the same sequence (34°) (Kostrhunova et al., 2010). Besides the nature of the platinated drug, the surrounding DNA sequence is also of major importance for helix stability. Indeed, monofunctional platinum adducts at 5'-TGC triplet induces major DNA destabilization (Brabec et al., 1992) but none at 5'-AGT or 5'-TGA triplet (Schwartz et al., 1989). DNA is not the unique nucleic acid destabilized by platinated derivatives as evidenced using *cis*-[PtCl(NH3)2(OH2)]+, *cis*-[PtCl(NH3)(c-C6H11NH2)(OH2)]+ and *trans*-[PtCl(NH3)(quinoline)(OH2)]+ (Figure 3) which not only destabilize ds-DNA but

**2.2.1 Cisplatin and other transition-metal antitumor agents as DNA destabilizing** 

also ds-RNA (Tm of -11°C and -5°C, respectively) (Hägerlöf et al., 2006).

Besides platinum derivatives, ruthenium compounds were developed as anti-cancer drugs. NAMI-A was the first ruthenium derivative that entered phase I clinical trials in 1999 as an anti-metastatic drug (Bergamo et al., 2002), followed by KP1019 (FFC14A) in 2003 (Hartinger et al., 2008). Two gallium compounds, gallium maltolate and KP46 (FFC11), also entered phase I clinical trials in 2003 (Lum et al., 2003). As for cisplatin, ruthenium derivatives evidenced DNA destabilization properties. This is particularly well described for Ru-CYM ([(6--cymene)Ru(II)(en)-(Cl)]+ and Ru-BIP, Ru-DHA or Ru-THA as biphenyl, dihydroanthracene or tetrahydroanthracene derivatives, respectively (Figure 3). Such organometallic ruthenium(II) arene complexes were rationally designed for chemotherapy with the idea that changing platinum for ruthenium would provide additional coordination sites in the octahedral complexes to modify the oxidation rate and change ligand affinity

**drugs** 

and binding kinetics (C.X. Zhang & Lippard, 2003). From this series, Ru-CYM presents the highest DNA helix destabilization activity, together with the smaller unwinding angle in supercoiled plasmid DNA (7° *vs*. 14° for Ru-BIP, Ru-DHA and Ru-THA), in correlation with its lack of intercalation and the formation of monoadducts at N7-dG (Nováková et al., 2009). New Ru-derivatives monodentate-Ru(II) and [Ru(terpy)(4,4'-(COLysCONH2)2bpy)Cl]3+ also destabilize DNA (Nováková et al., 2010; Triantafillidi et al., 2011). For gallium-complexed compounds, interaction of trivalent Ga-cations with calf-thymus DNA resulted in major helix destabilization with perturbations at A-T base pairs sites (R. Ahmad et al. 1996).

Fig. 3. Structure of cisplatin and other transition-metal agents as DNA destabilizing drugs and 3D orientation [mmdbId:47796] (cisplatin) and [mmdbId:69361] (oxaliplatin).
