Abstract:
Many widely used anticancer drugs such as m-AMSA act by inhibiting the action of the enzyme Topoisomerase II. They appear to act by stabilizing a key intermediate in the reaction cycle of the enzyme and forming a ternary complex of drug, DNA and enzyme, which ultimately results in the formation of lethal double-strand breaks in the DNA. Despite extensive experimental investigation, the structural details and the mechanism of formation of the ternary complex are not known. However indirect evidence concerning the structure and formation of the ternary complex is available from the many QSAR studies which have been carried out on anticancer drugs such as m-AMSA. A further complication is that while many of these drugs bind to DNA by intercalation, the relevance of this is unclear because it is not known whether the enzyme recognizes and binds to a pre-existing drug-DNA binary complex or whether the drug binds to a complex formed by the enzyme and DNA.
In the 9-anilinoacridine series of anticancer compounds, which includes m-AMSA, QSAR studies have shown that small changes in the molecular structure can lead to large changes in cytotoxic activity. An unexplained finding from these QSAR studies is why the close positional analogue of m-AMSA, o-AMSA, is much less effective in generating ternary complexes. Another observation which may be relevant to the difference in cytotoxic activity of the two compounds is that the binding of m-AMSA to DNA appears to be primarily enthalpy-driven while that of o-AMSA is driven primarily by entropic factors. A final observation to be explained is why m-AMSA appears to stimulate DNA cleavage by Topoisomerase II at sites in DNA adjacent to thymine or adenine residues. Other important questions to be answered concerning the mechanism of action of m-AMSA include whether or not it binds to specific sequences in DNA and whether there is a preference for binding with the phenyl ring of the drug in the major or minor groove. These observations motivated a molecular mechanics study of the binding of m-AMSA, o-AMSA and some other close analogues to DNA using the AMBER 3.0 force field. All calculations were carried out in vacuo.
The reversible binding of low molecular weight compounds to DNA is discussed in Chapter l. This is followed by a review of some previously published force field calculations and quantum mechanics calculations on the intercalation complexes of various compounds with DNA. The molecular mechanics method itself, is discussed in Chapter 2. This includes a discussion of force fields and their derivation, the techniques of potential energy minimization and molecular dynamics simulation and their respective advantages and disadvantages.
In order to understand simpler molecular systems, potential energy minimization calculations and dynamical simulations were carried out on oligonucleotides without intercalated drugs, and these are discussed in Chapter 3 and Chapter 4, Potential energy minimization calculations were carried out on double helical B-DNA dinucleotides and A- and B-DNA hexanucleotides in Chapter 3. The resulting conformations showed some differences from previously reported calculations on these sequences using earlier versions of the AMBER force field. The major difference was in greater repuckering of sugar residues away from the values in canonical A- and B-DNA. Calculations on the relative energies of A-DNA and B-DNA dinucleotides and hexanucleotides with different sequences were in general agreement with previously reported calculations with some differences in details. The conformation resulting from a potential energy minimization calculation on the B-DNA dodecanucleotide d(CGCGAATTCGCG)2 was in good agreement with an earlier calculation on this sequence using a different force field. In vacuo molecular dynamics simulations were carried out on the B-DNA sequence d(CGCGA).d(TCGCG) both with, and without hydrated counterions and are discussed in Chapter 4. The simulations were in general agreement with a previously reported simulation on this sequence, with some differences in details.
The intercalative binding of the unprotonated and protonated forms of m-AMSA to the double-stranded hexanucleotide d(TACGTA)2 was investigated in Chapter 5 in order to provide a comparison with similar calculations reported previously. For both species, a small number of distinct minima within 5 kcal/mol of the lowest energy conformations were found.
The unprotonated form of m-AMSA had a slight preference for intercalation with its phenyl ring located in the major groove rather than the minor groove. However the protonated form of m-AMSA, which is thought to be the biologically important form, strongly favoured intercalation with the phenyl ring in the minor groove rather than the major groove, mainly because of more favourable interactions of the phenyl ring and methanesulfonamide moiety in the minor groove. The unprotonated form intercalated with the long axis of the acridine chromophore approximately parallel to the long axes of the adjacent CG base pairs but in the protonated form, the chromophore was nearly perpendicular to the adjacent base pairs which were markedly non-planar. The finding of a perpendicular mode of intercalation for the protonated species is in general agreement with previous force field and quantum mechanics calculations on the intercalation of m-AMSA in DNA.
Potential energy minimization calculations were carried out on the intercalation of m-AMSA, o-AMSA, AMSA and their respective analogues without the methanesulfonamide sidechain in the double stranded dodecanucleotides, d(GC)6, d(CG)6, d(AT)6 and d(TA)6 and these are described in Chapter 6. The main aim of these calculations was to seek an explanation for the experimental observation that the binding of m-AMSA to DNA is primarily driven by enthalpic factors while that of o-AMSA is driven primarily by entropy. A second aim was to determine if the binding of m-AMSA and its analogues to DNA is sequence-specific. With only one exception, the net binding energies favoured binding in the minor groove over the major groove, often by a large amount. This was due to more favourable intermolecular van der Waals and electrostatic interactions in the minor groove. In most of the minor groove complexes, in the lowest energy conformations, the long axis of the acridine moiety was intercalated in a perpendicular mode similar to that found in Chapter 5. The calculations are at variance with previously reported experimentally-determined binding measurements because the binding of o-AMSA was calculated to be enthalpically more favourable than that of m-AMSA. This was due, principally, to more extensive hydrogen bonding between o-AMSA and DNA compared to m-AMSA. The increase in experimentally-determined binding entropy of o-AMSA compared to m-AMSA could be explained by differences in the interactions of the methoxy groups of m-AMSA and o-AMSA with a phosphate group at the intercalation site, with the methoxy group of o-AMSA causing a greater degree of solvent displacement from this phosphate group. The preferred binding site for m-AMSA was the minor groove of d(CG)6.. The preferred binding sites for o-AMSA were the minor grooves of d(GC)6 and d(AT)6.
Potential energy minimization calculations on the intercalation of m-AMSA and o-AMSA in four different double stranded dodecanucleotides containing the bases, T, A, C or G in one of the S'-positions of the intercalation site are described in Chapter 7. One aim of these calculations was to seek an explanation for the experimental observation that m-AMSA preferentially stimulates cleavage by Topoisomerase II at sites in DNA which contain adenine or thymine at the 5'-terminus of the DNA cleavage site (known as the -l position). A second aim was to determine any differences in the intercalation complexes formed by m- and o- AMSA with the same sequences which might explain their differences in cytotoxic activity. As found in the calculations in Chapter 5 and 6, the net binding energies were generally more favourable for the binding of m-AMSA and o-AMSA in the minor groove than the major groove. This was mainly due to more favourable intermolecular interactions in the minor groove. In the lowest energy conformations of each minor groove complex, m-AMSA and o-AMSA adopted the perpendicular mode of intercalation as was found in the calculations described in Chapters 5 and 6. The binding of o-AMSA was generally more favourable than that of m-AMSA, in both the minor and major grooves. A striking difference between the two ligands was a favourable interaction between the methoxy group and the phosphate group of the intercalation site in the minor groove complexes with o-AMSA but not with m-AMSA similar to that found in Chapter 6. In terms of net binding energies, m-AMSA preferred binding to the sequences with A and G in the -l position. In terms of intermolecular energies, m-AMSA preferred binding to the sequence with T in the -l position, in partial agreement with the results from Topoisomerase II cleavage stimulation experiments. The intermolecular energy in this sequence was augmented by two hydrogen bonds between m-AMSA and DNA, compared to one in each of the other three sequences.
In vacuo molecular dynamics simulations of the intercalation complexes of m-AMSA and o-AMSA with the four dodecamers were carried out and numerous differences were found in the average conformations of the sugar-phosphate backbone, the helical parameters and the widths and depths of the DNA grooves. These differences occurred between complexes of m-AMSA with the four different sequences and between complexes of m-AMSA and o-AMSA with the same sequence. These differences could explain (a) differences in binding to, or cleavage by Topoisomerase II at each of the binary complexes formed by m-AMSA with the four dodecanucleotide sequences and (b) differences in binding to, or cleavage by Topoisomerase II in the complexes formed by m-AMSA and o-AMSA with the same sequence.
Molecular dynamics simulations of intercalated complexes containing a break at the scissile P-O3' bond, resulted in kinetically stable dynamical structures in which the average P…O3' distance was significantly smaller in the complexes with o-AMSA than the corresponding complexes with m-AMSA, suggesting that P…O3' religation by Topoisomerase II might be less hindered by o-AMSA. The methoxy group of o-AMSA, but not m-AMSA, had a relatively strong interaction with the scissile phosphate and might stabilize it such that the P…O3' distance is smaller than in the m-AMSA complex. In all complexes the sulfone oxygens of m-AMSA and o-AMSA formed hydrogen bonds with the hydrogen atom attached to the scissile 03' atom. This might also explain the ability of m-AMSA to hinder the religation reaction of Topoisomerase II.