Abstract 1630: C8-linked pyrrolobenzodiazepine (pbd)-benzofused conjugates with low-picomolar in vitro cytotoxicity
Authors: Mantaj Julia , Jackson Paul JM , Pepper Chris , Thurston David E. , Rahman Khondaker M. 
Published: May 2015

There is significant interest in developing small-molecule ligands that can locate and bind to specific sequences of DNA as potential antitumor agents. Such molecules could be used, for example, to inhibit transcription factors and other control proteins from interacting with DNA at their consensus recognition sites. A set of DNA-interactive benzofused building blocks based on phenyl-substituted heterocycles have been developed with sufficient length to span two DNA base pairs. These building blocks have been conjugated to DNA minor-groove covalent-binding pyrrolobenzodiazepine (PBD) molecules via a four-carbon linker to produce C8-linked PBD-Benzofused hybrid molecules. These focused libraries contained structural variations where the benzofused building blocks were either directly linked to the PBD core or separated by pyrrole/imidazole heterocycles or GC-recognizing methylpyrrolebenzenamine (MPB) biaryl building blocks. The design rationale was based on the unique longitudinal curvature of these PBD conjugates which produces greater isohelicity with the DNA minor groove, and also on predicted hydrogen-bonding interactions with individual DNA bases.

These molecules showed significant enhancement of melting temperatures of designed oligonucleotides in a FRET-based assay compared to the PBD core alone, highlighting the synergistic effect of joining non-covalent benzofused rings to the covalent-binding PBD unit. The cytotoxicity of these compounds was assessed in primary CLL cells and a panel of other tumor cell lines (A431, A2780, A549, MIA PaCa2, HeLa and MDA-MB-231) after 48h and 96h exposure using the Annexin V apoptosis assay and the MTT colorimetric assay, respectively. Normal lymphocytes and the non-tumor fibroblast cell line WI38 were used as controls. Some of these compounds were highly potent (i.e., low picomolar) in the primary CLL and other cell lines with at least 3-orders of magnitude selectivity compared to the normal lymphocytes or WI38 cells. Importantly, molecules with different distances of separation between the benzofused ring moieties had significant differences in cytotoxicity, thus not only providing useful SAR information but also suggesting that there was scope for optimizing activity through a medicinal chemistry approach.

The effect of these conjugates on regulation of the NF-κB-associated regulatory proteins IKKα and IKKβ was studied in the CLL and breast cancer cell lines in which NF-κB is known to be active and closely correlated with the initiation and progression of malignancy. The lead PBD-Benzofused conjugate KMR-28-32 exhibited marked inhibition of both IKKα and IKKβ proteins at 4h and 24h time points at concentrations of 0.1nM and 1nM, respectively, with negligible effect on tubulin expression which was used as a housekeeping control.

Citation Format: Julia Mantaj, Paul JM Jackson, Chris Pepper, David E. Thurston, Khondaker M. Rahman. C8-linked pyrrolobenzodiazepine (pbd)-benzofused conjugates with low-picomolar in vitro cytotoxicity. [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 1630. doi:10.1158/1538-7445.AM2014-1630