Interaction of SJG-136 with cognate sequences of oncogenic transcription factors.

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 them with the appropriate 3-dimensional shape 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 linkage with the C2-NH2 group of a guanine base. The PBD dimer SJG-136 is presently in Phase II clinical trials in ovarian cancer and leukaemia. Transcription factors act through direct or indirect binding to specific DNA consensus sequences within gene regulatory regions, or by forming a complex with other proteins which then act as transcription factors. Covalent or non-covalent interaction of small molecules with key consensus DNA bases can prevent a transcription factor from recognizing its cognate sequence, thus preventing expression of genes critical for the survival and proliferation of cancer cells. The cognate sequences of a number of oncogenic transcription factors are unusually rich in GC sequences and provide ideal alkylation substrates (for both inter- and intrastrand cross-links and mono-alkylation) for both PBD dimers (e.g. SJG-136) and PBD monomers, and there is growing evidence that PBDs exert their pharmacological effects through this mechanism. We have developed a reversed-phase ion pair HPLC/MS method as a tool to evaluate the interaction of DNA-binding molecules with oligonucleotides of varying length and sequences. Using this methodology along with custom-designed oligonucleotides containing the cognate sequences of the transcription factors NFκB, AP-1, EGR-1 and Stat3, all of which contain ideal GC-rich binding sequences for SJG-136, we have observed both kinetic and thermodynamic adduct formation with SJG-136. Crucially, we observed a significant difference in rate and extent of adduct formation between the different cognate sequences which could partly explain the differences in activity of SJG-136 toward different tumour cell lines. For example, SJG-136 formed an adduct rapidly with the NFκB sequence (>50% adduct within 3 hours), moderately with the STAT3 (>35% adduct within 3 hours) and EGR-1 cognate sequences (>30% adduct within 3 hours) while showing insignificant adduct formation with the AP-1 cognate sequence (< 5% adduct within 3 hours) even though their GC contents are comparable. These findings add to knowledge of the possible mechanism of SJG-136, and could help with interpretation of the activity of SJG-136 in various biochemical and pharmacological assays.