Rahman, Khondaker Mirazur; (2009) Design, Synthesis and Evaluation of DNA Interactive Small Molecules. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Most non-covalently binding DNA-interactive molecules based on pyrrole-type building blocks (e.g., polyamides such as netropsin) have a preference for AT-rich sequences. One objective of this project was to develop a set of novel biaryl building blocks with the potential to recognise GC sequences. Using solution phase combinatorial chemistry, a library of 135 novel polyamides was initially designed and synthesized containing a biaryl unit as a key feature. A Fluorescent Intercalator Displacement assay indicated that library members containing a 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif switched to a GC preference. Further evaluation using HPLC/MS, DNA thermal denaturation and footprinting assays confirmed this strong GC preference. Unexpectedly, the biaryl polyamides were also found to be highly selective for some G-quadruplex DNA structures, with related selective cytotoxicity in relevant cancer cell lines. The MPB biaryl motifs were conjugated to pyrrolobenzodiazepine (PBD) molecules to produce covalently-binding agents with extended GC-selectivity within duplex DNA. Many of these MPB-PBD conjugates exhibited potent bactericidal activity against MRSA and VRE clinical isolates, and PBD-Py-MPB and PBD-Im-MPB conjugates showed significant selective in vitro cytotoxicity in a number of tumour cell lines. Finally, the HPLC/MS methodology was further developed to evaluate interaction of the PBD dimer SJG-136 with duplex oligonucleotides of varying length and sequence. In addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, SJG-136 was shown to form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra-or interstrand cross-links are feasible. These observations impact on the proposed mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials. The HPLC assay was also used to assess the impact of microwave radiation on PBD-DNA interaction where it was found that microwave levels of insufficient power to cause heating led to a significant increase in the rate of reaction between PBDs and DNA. The HPLC method was also used to observe a dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex PBD-DNA adducts, and to study the reversibility of PBD-DNA adducts.

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