Narayanaswamy, Mathangi; (2009) An assay combining HPLC and MS to evaluate the DNA interaction of pyrrolobenzodiazepines (PBDs) with oligonucleotides. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Currently the main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA involves the electrophoresis of relatively long radiolabelled duplex DNA fragments (e.g., typically ~ 2000 base pairs) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading and a neutral gel allows re-annealing of the cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion-pair RPLC and mass spectrometry that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 base pairs). Advantages of this assay include rapid throughput compared to electrophoretic methods and the use of readily available short non-radiolabeled oligonucelotides of any sequence, thus facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by mass spectrometry. Using this new method we have investigated the rate of DNA cross-linking and sequence-selectivity of the interstrand cross-linking agent SJG-136 (SG2000, BN2629), a pyrrolobenzodiazepine (PBD) dimer currently in Phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by HPLC, and to allow positive identification of these individual species by mass spectrometry. A further benefit compared to electrophoretic methods is that kinetic information could be obtained and compared for specific binding sequences. The HPLC and MS assay was later applied to study the intrastrand cross-links and monoadducts formed by SJG-136. The data obtained from the assay demonstrated that the PBD dimer SJG-136 is capable of forming intrastrand cross-links and monoadducts in addition to the inter-strand crosslinks which, it was previously thought to from exclusively The HPLC and MS assay was then suitably modified to study the binding of the PBD conjugate GWL-78. Previous studies have shown that PBDs bind exclusively to double-stranded DNA with their A-ring oriented towards the 3'-end of the bound strand. We report here that PBD conjugate GWL-78 can form stable adducts with single-stranded l7-mer oligonucleotides. The HPLC and MS assay has confirmed their compositions and demonstrated that rate and extent of formation varies with base-pair sequence. Modelling studies suggest that, with C11(5)-stereochemistry and with the A- ring of GWL-78 oriented unusually towards the 5'-end of the covalently-modified guanine, a highly compact hairpin structure of low energy results. For 17-mer 5'-TATAAGAAAATCTTATA-3' (Seq-1), the reaction is rapid and quantitative. When GWL-78 is reacted with the equivalent annealed duplex structure 5'- TATAAGAAAATCTTATA-3'/5'-TATAAGATTTTCTTATA-3' (Seq-1/Seq-2), the two corresponding 1:1 adducts are still the main products along with small amounts of two 2:1 ligand/duplex adducts. This suggests that, despite annealing, Seq-1 and Seq-2 exist as an equilibrium mixture of hairpin, random coil and duplex species, with a predominance of the former. Trapping of the hairpin rather than induction is considered more likely, as it is well documented that PBDs require minor groove structure for covalent binding. This suggests a possible alternative explanation for the biological activity of PBDs.

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