Molecular Dynamics Studies of Changes in the DNA- Structure as Result of Interactions with Cisplatin

The 3’-monofunctional adduct of cisplatin and d(CTCTG*G*TCTC)2 duplex DNA in solvent with explicit counter ions and water molecules were subjected to MDsimulation with AMBER force field on a nanosecond time scale. In order to simulate the closure of the bond between the Pt and 5’-guanine-N7 atoms, the forces acting between them were gradually increased during MD. After 500-800 ps the transformation of the mono-adduct (straight DNA with the cisplatin residue linked to one guanine-N7) to the bus-adduct (bent DNA where Pt atom is connected through the N7 atoms of neighboring guanines) was observed. A cavity between palatinate guanines is formed and filled with solvent molecules. The rapid inclination of the center base pairs initiates a slow transition of the whole molecule from the linear to the bent conformation. After about 1000-1300 ps a stable structure was reached, which is very similar to the one described experimentally. The attractive force between the Ptatom and the N7 of the second guanine plays the main role in the large conformational changes induced by formation of the adduct-adduct. X-N-Pt-N-torsions accelerate the bending but a torsion force constant greater than 0.2 Kcal/mol lead to the breaking of the H-bonds within the base pairs. The present study is the first dynamical simulation that demonstrates in real time scale such a large conformational perturbation of DNA. Introduction Nearly all mechanisms of cisplatin cytotoxic activity proposed to date consider DNA to be the primary biological target [1]. Interactions with other cellular components have also been observed, but there is no convincing evidence that these are involved in the mechanism of drug action [2]. The major products of DNA-platination are 1,2-intrastrand crosslinks involving adjacent bases with cis-[Pt(NH3)2{d(GdG)}] (cis-GG) and cis-[Pt(NH3)2{d(AdG)}] (cis-AG) comprising 47-50% and 23-28% of the adducts formed respectively. The remaining 10-13% consist of mono-functional derivatives of guanine and products of 1,3-intrastrand cross-platination [3]. By the mid 90’s the kinetic pathways of the cisplatin-DNA interaction were elucidated [4]. In a hydrolysis reaction with a half-life of 2.3 h at 37 °C one chloride ligand in cisplatin is replaced by water [5]. The product of this partial hydrolysis cis-[Pt(NH3)2(H2O)Cl] reacts rapidly with DNA, forming a mono-adduct with either adenine or guanine. These monoadducts were postulated to be inactive in processes leading to the death of cancer cells [3] because other platinum compounds which bind to DNA only in monofunctional manner show no anticancer activity. Following the hydrolysis of the second Pt-Cl bond, the Gmonoadduct undergoes a rapid chelation reaction and forms a bis-adduct if the 5'-base adjacent to the guanine is A or G. This reaction perturbs the global structure of the DNAmolecule with respect to B-DNA so that a significant curvature around the platination site is induced [6]. The structures of these bis-adducts were elucidated in crystal by Xray diffraction [7,8] and in solution by NMR spectroscopy [9,10], respectively. Both results are noticeably different, presumably because the interaction between adjacent DNA molecules leads to significant deformations in the crystal. However, in both sets of data the H-bonds between cytosine and corresponding platinated guanine are conserved. One of the factors thought to determine the anticancer activity of cisplatin is the geometry of the adduct with DNA [3]. A major role is played by the extension of the width of the minor groove during the bending process, by which a nonpolar part of DNA becomes accessible for solvent water molecules. In addition, the increased width allows specific interactions with proteins that would not otherwise bind with high affinity to normal B-DNA, such as HMG-group proteins [11-15]. Prediction of the final structure for adducts of DNA with platinum complexes other than cisplatin will only be possible when dynamics of the bending mechanism are understood in detail. To date, such studies are lacking. The linear structure of DNA is stabilized in solution by significant stacking stabilization energies and hydrophobic interactions, but still the energy gain due to the formation of only one additional PtN-bond must be sufficient to favor the bent structure of the complex with cisplatin. Three mechanisms of chelation are conceivable: (i) the entire DNA molecule is permanently bent to some extent as a result of Brownian motion or interactions with DNA-binding proteins. As soon as such a deformation leads to an appropriate position of Pt and N7-atom of nonplatinated guanine and allows for covalent bond formation, further irreversible bending of the whole DNA structure will occur; (ii) two neighboring base pairs, one platinated and the other not, have some degrees of freedom so that they could incline with respect to each other due to thermal motion and reach a suitable position to form the bis-adduct; (iii) the attractive forces between Pt(II) and 5’-N7 result in an inclination of two base pairs with respect to each other, following formation of a new bond. The aim of this study was to understand the details of this bending process. 3’G-monoadduct as initial point was chosen comparing 3Dstructures of two possible monoadducts. Transformation of 5’monoadduct to bis-adduct seems to be less probable because the distance between potential reaction centers (Pt and N7) in 5’-monoadduct is about 5.5Å, what is greater than one in 3’monoadduct (3.6 Å). This assumption is also confirmed by the literature data [16]. AMBER is a standard force field used in DNA simulations. The versions up to 3.0 [17,18] treated the solvent as a continuum with adjustable dielectric constant [19]. As of AMBER 4.1 [20] solvent molecules can be taken into account explicitly. This was applied to DNA [21], RNA [22] and a copolymer [22]; in those simulations average helical parameters and their fluctuations were measured. With the AMBER force-field the canonical B-DNA structure in water solution can be well reproduced for several nanoseconds [2326]. Geometry optimization has been done for platinum complexes with small ligands [27-29] such as two bases, and force field parameters for the bonds with Pt(II) have been derived. Molecular dynamics simulations of the cisplatinDNA complex with ten base pairs were performed by Scheeff et al. [30] in vacuo. As electrostatic interactions are overestimated without water, the authors had to decrease the charge for phosphate groups and applied atomic constraints to tie the base pairs on both ends of oligomer in order to keep both DNA strands together. This simulation resulted in a stable trajectory for the bent structure. No realistic simulation of the bending process itself is possible, however, without taking into account the hydrodynamics of the solvent. Computational Details For molecular dynamic (MD) simulation the AMBER 5.0 software package [31] has been used which includes version 4.1 of the AMBER force field [20]. The AMBER 3.0 force field has been extended to Pt(II) compounds in [32]. We transferred these additional parameters to AMBER 4.1 without modification. The partial charges for the atoms in the guanines are significantly modified by the platination due to electron withdrawal to the positively charged platinum atom (tab 1). We applied the difference between platinated and free guanines described in [32] to the atomic charges from AMBER 4.1. A similar charge transfer technique has been used before in force field development [33,34]. We generated a DNA d(CTCTGGTCTC)2 duplex using the NUCGEN script included in the AMBER 5.0 package. The length was limited to 10 base pairs rather than 12 in the experiments in order to speed up calculations. The monoadduct of cisplatin and DNA was created by connecting a Pt(NH3)2 residue to the N7 atom of the 3 ́ guanine, the Pt lying in the guanine plane. All parameters for this Pt-N7 bond were set to their normal values. The resulting distance between Pt and N7 in the second guanine was about 3.6 Å, which is almost twice the bond length. The cisplatin-DNA complex was surrounded with a solvent bath, having rectangular shape (38x41x51 Å 3 ) and consisting of approximately 3500 TIP3P [35] water molecules. Each strand of the duplex contains 9 negative charges located on the phosphate groups and the cisplatin residue has an electric charge of +2. 16 Na+ ions were added providing electroneutrality of the entire system. They were placed around the solute molecule calculating a Coulomb potential grid with LEaP program in the AMBER package. This procedure finds appropriate positions for Na+ ions by minimizing the electrostatic energy of the system. The solvent box provides 8 Å minimum distance between DNAatoms and the walls of the box in Yand Zand 10 Å in Xdirection allowing some additional space for bending of DNA chain in the YZ-plane. According to the AMBER MD protocol, the rotational and translational motions of the solute molecule are regularly removed and the appropriate position of the DNA-adduct in the anisotropic box is provided during the entire MD-trajectory. The equilibration of the system is described elsewhere [25] in detail. MD simulations were performed in the NTP-ensemble with a 2 fs time step. During the equilibration stage the volume was adjusted so that the pressure in the system was approximately 1 atm. The Berendsen algorithm for temperature bath coupling [36] was utilized to keep the temperature of the core reasonable close to that of solvent. Table 1. Atomic charges