Abstract 3696: Molecular dynamics simulations of C7/C7′-linked pyrrolobenzodiazepine (PBD) dimers
Authors: Jackson Paul J. M. , Rahman Khondaker Miraz , Thurston David E. 
Published: August 2015

Abstract
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of naturally occurring and synthetic DNA minor-groove binding agents (MGBAs) characterised by their ability to covalently bind to the C2-amino groups of guanine bases. Although natural PBD monomers have low micromolar cytotoxicity, synthetic PBD dimers have garnered significant attention due to their potent cytotoxicity and use in antibody-drug conjugates (ADCs). The cytotoxic nature of PBD dimers is thought to relate to their ability to form inter- and intrastrand cross-links in DNA. The first known PBD dimer had its two PBD units linked through their C7/C7′-positions, and was reported by Suggs and co-workers. This molecule was shown to reversibly cross-link DNA, a feature that may account for its poor cytotoxicity relative to C8-linked dimers which are used as ADC payloads. To investigate this, we have undertaken Molecular Dynamics (MD) studies on the interaction of C7-dimers with DNA and compared results to those for C8-linked PBD dimers. This has led us to propose an alternative mechanism to explain the discrepancy in potencies as described below.
MD simulations were conducted over 10ns in explicit solvent. In this study, Suggs’ published C7/C7′-linked dimer and an equivalent C8/C8′ linked structure were first compared, and this was followed by comparison of the known C8-linked PBD dimer SG-2202 and an equivalent C7/C7′-linked version. SG-2202 is known to preferentially bind to the sequence 5′-pu-G-A-A/T-A/T-C-py-3′. As each of the PBD dimers analysed have an equivalent central linker (i.e., five methylenes), each of the structures were simulated in the sequence 5′-GCGATTCTCGC-3′. The results suggested that C8-linked dimers interact with DNA to a far greater extent than C7-linked dimers, a feature attributable to the level of curvature (“isohelicity”) of C8-linked dimers, and this was supported by free energy of binding calculations. Crucially, the simulations showed that the C7-linked dimer family does not form stabilising non-covalent interactions with the DNA minor-groove as previously thought. Furthermore, due to its poor isohelicity, simulations predict that only a single alkylation event takes place initially, and this is followed by the second alkylation which is dependent on DNA breathing to align the second PBD unit with an appropriate guanine. Simulations suggested that the C7-position of any PBD has two potential orientations: (1) bulky groups such as a sugar (i.e., as in sibiromycin) point out of the minor groove, and (2) smaller substituents (e.g., a methylene linker) point along the minor groove floor. The latter explains why it is possible for C7-methylene-linked PBD dimers to cross-link DNA albeit with less efficiency compared to C8-linked dimers. Studies are now underway to analyse the curvature of PBD-type molecules to develop tools (e.g., QSAR) for the correlation of shape and cytotoxicity to facilitate the discovery of MGBAs with more potent cytotoxicity.
Citation Format: Paul J. M. Jackson, Khondaker Miraz Rahman, David E. Thurston. Molecular dynamics simulations of C7/C7′-linked pyrrolobenzodiazepine (PBD) dimers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2015-3696