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Title: Elucidation of the sequence selective binding mode of the DNA minor groove binders SJG-136 and adozelesin, by high field ¹H NMR and restrained molecular dynamics
Author: Hopton, Suzanne
ISNI:       0000 0001 3581 6067
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2007
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The binding of two covalent minor groove binding ligands to duplex DNA has been investigated using high field IH NMR and NMRINOE rermed molecular modeling techniques. Various duplex DNA strands were synthesised and then reacted with the covalent minor groove binding drugs SJG-136 and adozelesin. The drug-DNA adducts were analysed using 2D NMR techniques and the resulting data used to produce NOE distance refined molecular models. SJG-136 is a synthetic dimeric covalent minor groove binder based on the PBD family ofantitumour antibiotics. These ligands are isolated from various Streptomyces species and exhibit potent in-vitro and in-vivo activity. They are known to form inter-strand cross-links with duplex DNA by reaction with the exo-cyclic NH2 group of guanine. Adozelesin is a covalent minor groove binding analogue of the CPI antitumour antibiotic (+)-CC-I065. The CPIs are known to alkylate duplex DNA by reaction with the N3 position of an adenine base, and owe their biological activity to an ability to block DNA replication. Four novel ligand-DNA adducts have been produced and fully assigned using sequential assignment techniques. The NMR data collected has been used to confinn the sites of alkylation, orientat,i.on of the ligand residue and stereochemistry of the interactions. NOE distances are then used to produce accurate NMR refined molecular mode!s of each adduct. The 5'd(CICGATCICG)2-SJG-136 adduct was found to bind covalently to exo-cyclic NH2 groups on guanine bases of opposite DNA strands - forming an inter-strand crosslink separated by 4 base pairs. Stereochemistry was assigned as'S' at both reaction sites and self complementarity and p-helical structure of the DNA duplex was maintained. Molecular models show minimal distortion throughout the duplex, with only a small localised area of distortion in the central base pairs as a result of drug binding. The 5'd(CTCATCAC).(GTGATGAG)-SJG-136 adduct has been successfully produced, with alkylation sites confirmed as the exo-cyclic NH2 groups of two guanine bases on the same DNA strand. This represents the first identification of an intra-strand cross-linked PBD adduct, generating an. exciting new possibility involving targeting of the human telomere repeat sequence using PBD type ligands. Once again stereochemistry at the reaction sites is confmned to be '8' in both cases and the drug is found, as expected, to associate more closely with the modified DNA strand than with its complementary sequence. A 5'd(CGATTAATCGh-adozelesin mixed adduct has been generated, and found to contain a mixture of adducts in an approximately SO/50 ratio. One adduct has retained Watson-Crick base pairing within the DNA duplex, while in the second the central AT step had adopted a Hoogsteen confonnation. In both adducts a significant overlap and stacking of tJ1e benzofuran subunits is observed. The discovery of novel 'stacked' adducts for CPI ligands suggests an alternative model for minor groove drug interactions and similarities can be drawn between the adozelesin stacked molecules and analogous stacking in the non-covalent minor groove binding lexitropsin drugs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available