Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures.

As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C selectivity is thought to relate to the wider minor groove in G/C regions and the presence of the exocyclic 2-amino groups of guanines which project into the minor groove and prevent multiple close van der Waals contacts with the G/C-rich minor groove. AT-selective structures such as distamycin and netropsin orientate in the minor groove in an “NH down” conformation, with their NH groups pointing into the minor groove to enhance the isohelicity of the structures and produce favourable van der Waal interactions with the groove floor below. Using a distamycin scaffold as a starting point we introduced biaryl building blocks in place of pyrroles to change the curvature and van der Waals interaction potential of this novel class of polyamides. We started by designing a library of fragments to span two DNA base pairs based on simple phenyl-substituted heterocycles of sufficient length. With each library member containing one heterocyclic and one carbocyclic component, sufficient diversification was achieved through modifications to, and substitutions within, both components. This allowed the production of libraries of building blocks possessing different curvatures and hydrogen-bonding abilities with the potential to complement functional groups and hydrogen bond donors and acceptors in the walls and floor of the minor groove. A fluorescent intercalator displacement (FID) assay was then used to select molecules with a preference for GC-rather than AT-rich sequences which identified the 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif. Molecular dynamics simulations were undertaken using the AMBER v11 software in implicit solvent over a time-scale of 20ns with the structures in both a “carbonyl-in” and “NH-in” conformation. This demonstrated that the MPB component within the biaryl polyamides undergoes a switch in orientation, with a “carbonyl down” conformation preferred which induces GC specificity. As well as occupying an idealised hydrogen-bonding distance between two side-by-side guanine residues, MPB units contained within the MPB biaryl polyamide structures showed superior hydrogen bonding interactions with GC compared to AT sequences, with each carbonyl of the MPB unit hydrogen bonding to a guanine residue. This was further supported by free-energy of binding calculations undertaken using AMBER-MMPBSA, where the MPB units showed a distinct preference for GC sequences (a difference of -13kcal/mol in favour of “carbonyl -in” over “NH-in” structures). This discovery has been applied to the design and synthesis of more-complex MPB-containing DNA minor-groove binding ligands with a high level of GC-selectivity and significant in vitro and in vivo antitumour activity.