Abstract 5329: Molecular dynamics simulations of sibiromycin suggest a role for the c7-sugar in transcription factor inhibition
Authors: Jackson Paul J M , Rahman Khondaker M. , Thurston David E. 
Published: May 2015

Abstract
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are naturally occurring DNA minor-groove binders characterised by their ability to covalently bind to the C2-amino groups of guanine residues. Although the PBDs have various C2- and A-ring substituents, one member, sibiromycin, has a C7-glycosyl (sibirosamine) residue. It is the most cytotoxic member of the PBD monomer family, and has the highest DNA-binding affinity. The extreme potency was originally ascribed to a combination of hydrogen bonding interactions between the C7-sibirosamine and DNA, and improved cellular penetration due to the sugar. However, we have now carried out Molecular Dynamics (MD) simulations on the interaction of sibiromycin with DNA, which has led us to propose an alternative mechanism to explain its potency.
Implicit solvent (25ns) and extensive explicit solvent simulations were undertaken to investigate the sequence-selectivity of sibiromycin, and to observe the role of the C7-sugar in DNA-binding. The results suggested that sibiromycin has a preference for the sequence 5′-X-G-G/C-3′, compared to 5′-Pu-G-Pu-3′ for other PBD monomers (Pu = purine, X = any base). Crucially, the simulations showed that the C7-sugar does not form stabilising hydrogen bonds with the DNA minor-groove walls or floor as previously thought, but instead points out of the groove orthogonally to the groove floor. Furthermore, the simulations suggested that the C7-position of any PBD has two potential orientations: (1) large bulky groups such as sugars point out of the minor groove, (2) smaller substituents such as a methylene linker points along the floor of the minor groove. The latter is exemplified by the ability of the C7-methylene-linked PBD dimers to cross-link DNA.
Our observations on the orientation of C7-substituents prompted us to question whether the C7-sugar of sibiromycin could protrude sufficiently from the minor groove to inhibit the approach of a DNA-binding protein, thus accounting for its superior cytotoxicity. Based on a previous report that sibiromycin could displace a peptide from a 19-bp oligonucleotide corresponding to the consensus sequence for the transcription factor GAL4, we chose this as a suitable system for further studies. Thus we undertook a docking study with the crystal structure of GAL4 bound to its consensus DNA sequence, with sibiromycin docked at every potentially reacting guanine. We observed significant steric interaction between the sibiromycin C7-sugar and Arg46 of the homodimer B of GAL4, a process that could contribute significantly to its cytotoxicity.
These studies have suggested a unique alternative mechanism of action for sibiromycin. Studies are underway in our laboratory to explore this, and if proved correct, novel inhibitors could be designed based on PBD molecules targeted to the consensus binding sites of individual transcription factors, using a bulky group at the C7-position to sterically hinder protein approach.
Note: This abstract was not presented at the meeting.
Citation Format: Paul J M Jackson, Khondaker M. Rahman, David E. Thurston. Molecular dynamics simulations of sibiromycin suggest a role for the c7-sugar in transcription factor inhibition. [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 5329. doi:10.1158/1538-7445.AM2014-5329