Abstract 5370: Use of molecular dynamics simulations to rationalise the DNA sequence-selectivity of pyrrolobenzodiazepine-MPB conjugates
Authors: Jackson Paul JM , Rahman Khondaker M. , Thurston David E. 
Published: May 2015

Abstract
We have previously reported the identification of a GC-interactive non-covalent-binding moiety, a biaryl 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif, through a combined approach involving rational library design and a Fluorescent Intercalation Displacement (FID) assay. This MPB motif, when tethered to a covalently-binding pyrrolobenzodiazepine (PBD) moiety via either an imidazole or pyrrole linkage to form PBD-Imidazole-MPB or PBD-Pyrrole-MPB structures, results in cytotoxicity in the low picomolar range in some tumour cell lines. To assess the DNA-binding affinity and sequence selectivity of PBD-Imidazole-MPB (TSG-1301) and PBD-Pyrrole-MPB (TSG-1302), we have previously reported the use of a FRET-based DNA melting assay based on a duel-labelled hairpin-forming oligonucleotide containing only one PBD binding site. Although having similar cytotoxicities, the sequence selectivity of TSG-1301 and TSG-1302 differ markedly. Melting temperatures (ΔTm) showed that TSG-1301 stabilised both AT- and GC-rich sequences of DNA to an equal extent (i.e., ΔTm of 6°C for each), with TSG-1302 selectively stabilising AT-rich sequences with an approximately 3.5-fold selectivity (i.e., ΔTm of 5°C for AT-rich sequences and 1.4°C for GC-rich). Therefore, Molecular Dynamics (MD) simulations were undertaken to try to understand these differences in sequence selectivity.
Simulations were first conducted in implicit solvent for 25ns, and confirmed using extensive explicit solvent simulations using the AMBER software. The results clearly demonstrate the importance of the imidazole ring nitrogen of TSG-1301 in inducing GC-selective interactions through hydrogen bonding. Both the distance between the imidazole ring nitrogen and the terminal ester (an average of 12Å over the duration of simulation), and the shape of the molecule, were found to be crucial for the production of GC interactions, with the Im-MPB moiety ideally spanning side-by-side guanine residues. The AT-selectivity of TSG-1302 could be rationalised using similar techniques, with the Py-MPB moiety contributing to van der Waals interactions between the ligand and AT-rich DNA, due to the MPB moiety presenting a strongly van der Waals-interactive face to the minor-groove. These results were further exemplified using free energy of binding calculations from implicit solvent simulations which provided a difference of -2kcal/mol in favour of the interaction of TSG-1302 with AT-rich compared to GC-rich sequences. The results of this MD study are consistent with observations from our previously reported FRET studies, and the simulations have provided valuable insights into the mode of DNA-interaction of minor groove-binding ligands of this type. This information can be used for the rational design of further generations of agents with improved DNA sequence-selectivity.
Citation Format: Paul JM Jackson, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamics simulations to rationalise the DNA sequence-selectivity of pyrrolobenzodiazepine-MPB conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2014-5370