Abstract
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. They have a chiral centre at their C11a(S)-position which provides an appropriate three-dimensional shape for them to fit securely within the DNA minor-groove. They also possess a “soft” electrophilic imine moiety at their N10-C11 position which can form an aminal bond between their C11-position and the C2-NH2 group of a guanine base only when the molecule is secure within the minor groove. The PBD dimer SJG-136 has currently reached Phase II clinical trials in ovarian cancer and leukaemia in the UK and the USA, respectively. More recently, PBD dimer analogues are being attached to tumour-targeting antibodies to create Antibody-Drug Conjugates (ADCs), some which are now in early clinical trials with many others in pre-clinical development.Transcription factors (TFs) are sequence-specific DNA-binding proteins that bind to consensus DNA sequences, thereby controlling transcription. TFs regulate processes such as cell differentiation, proliferation and apoptosis. The interaction of a small-molecule with the consensus DNA sequences of TFs can prevent a TF from recognising its cognate sequence, thereby inhibiting the expression of genes critical for the survival and proliferation of cancer cells. The cognate sequences of a number of oncogenic TFs are unusually rich in GC sequences and provide ideal alkylation substrates (both cross-link and mono-alkylation modes) for both PBD dimers (e.g., SJG-136) and monomers (e.g., GWL-78 and KMR-28-39). There is growing evidence that PBDs exert their pharmacological effect through TF inhibition in addition to the arrest of the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. For this reason, there is now interest in using PBDs as the basis for a small-molecule strategy to target specific DNA sequences for TF inhibition as a novel anticancer therapy.
This project initially involved the application of a reversed-phase HPLC/MS method as a tool to evaluate the interaction of DNA-binding PBD molecules with oligonucleotides of varying lengths and sequences. Using custom oligonucleotides containing the cognate sequences of NF-B, EGR-1, AP-1 and STAT3 transcription factors, all of which contain ideal GC-rich binding sequences for the PBD dimer SJG-136, the HPLC/MS method was used to provide information on both kinetic and thermodynamic adduct formation. Interestingly, a significant difference in the rate and extent of adduct formation between the different cognate sequences was observed which may explain the differences in activity of SJG-136 in the various tumour cell lines. Furthermore, SJG-136 was found to form different types of adducts with the TF sequences studied indicating a preference for guanine binding sites within the individual sequences. In particular, the fastest reaction was observed with the AP-1 sequence (i.e., 100% adduct formation, and only one type of adduct formed) which led to guanine/inosine replacement studies to establish the precise DNA-binding site of SJG-136. During this study, the ability of PBD monomers (e.g., GWL-78) and dimers (e.g., SJG-136) to bond to a terminal guanine was demonstrated for the first time.
Further cellular experiments, the Polymerase Chain Reaction (PCR) and Western blotting, were carried out to support the observations made during the biophysical evaluation of SJG-136 with the NF-B, EGR-1, AP-1 and STAT3 sequences. In these studies, SJG-136 was shown to significantly down-regulate a number of AP-1- and STAT3-dependent genes in the human colon carcinoma cell line HT-29 and the human breast cancer cell line MDA-MB-231.
Overall, the findings in this project have added significantly to the knowledge of the mechanism of action of SJG-136 and related PBD compounds, and could be important for the correct interpretation of the results of future pre-clinical and clinical studies of molecules of this type.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Miraz Rahman (Supervisor) & David Thurston (Supervisor) |