Abstract 2321: Pyrrolobenzodiazepine (PBD) antitumour agents as chemical probes to determine the number of base pairs required for DNA minor groove structure

Abstract

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective covalent-binding DNA-interactive agents that fit perfectly in the minor groove of double-stranded DNA due to their isohelical 3-dimensional shape. They possess an electrophilic N10-C11 imine (or the equivalent) that can form a covalent aminal bond between their C11-position and the C2-NH2 group of a guanine. One synthetic PBD dimer, SJG-136, has just entered Phase II clinical trials. Previous studies using HPLC/MS, CD spectroscopy, high-field NMR, X-ray crystallography and gel-based assays such as DNA footprinting and in vitro transcription stop are all consistent with the requirement for duplex DNA for covalent binding. Conversely, it is well-documented that PBDs will not interact with single stranded DNA which lacks minor groove structure. In this study, these observations served as a basis to use PBDs as chemical probes to determine the minimum number of bases required to create a minor groove environment within a DNA duplex. Thus we studied reaction of the naturally-occurring anthramycin (3 base pair span) and the synthetic C8-linked PBD bis-pyrrole conjugate GWL-78 (6 base pair span) with a range of designed duplex oligonucleotides of increasing length (3 to 11 base pairs), each containing an AGA binding triplet. Reaction was monitored by HPLC/MS which provided data on both the rate and extent of reaction, and the structure of DNA adducts formed. For both anthramycin and GWL-78, we found that a minimum of 7 DNA base pairs were required for reaction to occur, suggesting that this may represent the minimum necessary to provide a suitable minor groove environment. Furthermore, we found that the rate of reaction for both PBDs increased with the length of the oligonucleotide between 7 to 10 base pairs. It is noteworthy that GWL-78 is approximately twice the length of anthramycin but both require at least 7 base pairs in order to covalently bind. This suggests that both may be responding to the formation of a minor groove environment in an oligonucleotide that reaches 7 base pairs in length, rather than a direct relationship between the length of the PBD and the number of base pairs in the oligonucleotide. These observations were supported by molecular modeling which confirmed that 7 base pairs are required to provide minor groove structure. This is the first study to use a chemical probe of this type to monitor formation of a minor groove. This information should be useful in the design of future generations of PBDs and similar DNA-interacting anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2011-2321