Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

Abstract

The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5'-d(TAATAACGGATTATT)·5'-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis), Zalypsis and PM01183. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink.

Figure 1.

(A) Chemical structures of the N-protonated forms of the drugs used in the melting simulations. In the case of the tetrahydroisoquinolines, dehydration of the hemiaminal (carbinolamine) yields the reactive iminium intermediate that reacts with the 2-amino group of a guanine (7). In the case of MMC, two-electron reduction of the quinone ring facilitates methoxide elimination, formation of a leuco-aziridinomitosene and opening of the aziridine ring to provide the initial alkylating agent that leads to a DNA monoadduct with the 2-amino group of a guanine; the second alkylating centre is the iminium ion that forms upon the reverse Michael elimination of carbamic acid from the monoadduct and undergoes a nucleophilic attack by the 2-amino group of the guanine in the opposite strand at a CpG step (6). (B) Schematic of the binding modes of the tetrahydroisoquinolines Yondelis®, Zalypsis® and PM-01183® (THIQ, left) and MMC (right) to the central CGG triplet of the DNA 15-mer studied. Covalent and hydrogen bonds are shown, respectively, as solid and dotted lines. Base numbers refer to their position in the 15-mer: strand bearing the adduct = 1–15; complementary strand = 16–30.

Figure 2.

Representative snapshots showing the time evolution of the DNA duplexes during the MD simulations in the absence of any bonded drug (top) and in the presence of Yondelis®, Zalypsis® or PM01183® covalently bonded to the blue strand, or MMC (bottom) covalently bonded to both blue and red strands. Drug carbon atoms are coloured in green. For more detailed visualization see the video in Supplementary Data.

Figure 3.

Fraction of the MD simulation time (measured at 10, 50, 100, 150 and 200 ns) during which individual base pairs remained WC hydrogen bonded in the drug-free DNA 15-mer of sequence 5′-d(ATTATTCGGTAATAA)·5′-d(TTATTACCGAATAAT). Hydrogen bonds were rapidly lost for those base pairs found at the strand termini whereas those making up the central CGG triplet were the most durable. Note the asymmetry in the melting profiles of the DNA stretches located 5′ and 3′ to the central CGG triplet. This profile is to be compared with that obtained when these TAA and ATT stretches were swapped (Supplementary Figure S2).

Figure 4.

DNA base-pair disruption along the production phase of the MD simulations of the free and drug-bonded DNA 15-mers. All the original base pairs were progressively lost in the drug-free DNA, the last ones belonging to the central CGG triplet. In all the drug-bonded complexes, however, this triplet remained H-bonded at the end of the simulation. Furthermore, in the DNA molecules containing the bonded tetrahydroisoquinolines, the WC base-paired region was extended further due to the occurrence of some conspicuous reannealing events (marked by a vertical arrow), which were more marked for Zalypsis® and Yondelis® than for PM01183®.

Figure 5.

Fraction of total MD simulation time during which a particular base pair was disrupted in the DNA 15-mer (note that for drug-free DNA this plot is the reverse of that shown in Figure 3). The lower frequency of the unpaired state in the central CGG triplet was extended to the ATT sequence 3′ to it (i.e. A10:T21 and T11:A20 base pairs) in the duplexes containing bonded Yondelis® and Zalypsis® (vertical double-headed arrow) as a consequence of the reannealing events shown in Figure 4.

Figure 6.

Distances between the ring centroids of MMC and different DNA nucleobases during the simulated melting process. The drug’s chromophore (CPK spheres coloured in orange) served as a platform for hydrophobic contacts with different nucleobases from one of the melted strands (that coloured in red), in the chronological order shown at the top of the figure. The dotted line given as a reference at 3.4 Å represents the canonical distance between stacked nucleobases in a standard B-DNA double helix.