Effect of hairpin loop structure on reactivity, sequence preference and adduct orientation of a DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour agent.
The pyrrolobenzodiazepines (PBDs) are a family of covalent-binding DNA-interactive minor-groove binding agents with a thermodynamic preference for binding to 5'-Pu-G-Pu-3' sequences (Pu = Purine) but a kinetic preference for 5'-Py-G-Py-3' (Py = Pyrimidine). Using HPLC/MS methodology and a range of designed hairpin-forming oligonucleotides, the kinetics of reaction of a C8-bis-pyrrole pyrrolobenzodiazepine (PBD) conjugate (GWL-78, 2) with sixteen isomeric oligonucleotides has been evaluated, each containing a single PBD binding site in one of two locations. The PBD-binding base-pair triplets were designed to include every possible combination of A and T bases adjacent to the covalently-reacting guanine, with the set of hairpins consisting of isomeric pairs containing the same sequence in the hairpin stem but with either hexaethylene glycol (HEG) or TTT loops. The PBD 2 reacted most rapidly with TGT and TGA sequences, with the possibility that adducts might form in both the 3'- and 5'-directions with some sequences according to modelling studies. A faster reaction rate was observed for all hairpins containing the HEG loop except one (Seq 10) when the PBD binding triplets were located either near the loop or adjacent to the 5'-end. Modelling studies have suggested that this difference in reactivity could be due to the structural flexibility of the HEG loop allowing both A-ring-3' and A-ring-5' adducts to form, while a TTT loop should favour only A-ring-5' adducts due to steric considerations. These findings contrast with the results reported by Nguyen and Wilson for the interaction of non-covalent DNA-binding molecules with DNA hairpins, where the loop structure was found to have little effect on interaction in the main stem of the hairpin.
Institute of Pharmaceutical Science, Britannia House, 7 Trinity Street, London SE1 1DB, UK. david.thurston@kcl.ac.uk k.miraz.rahman@kcl.ac.uk.
David E
DE
Thurston
Higia
H
Vassoler
Paul J M
PJ
Jackson
Colin H
CH
James
Khondaker M
KM
Rahman
D000970:Antineoplastic Agents / Q000378:metabolism*; D020029:Base Pairing; D001483:Base Sequence; D001569:Benzodiazepines / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism*; D055029:Inverted Repeat Sequences; D008958:Models, Molecular
D000970:Antineoplastic Agents; D018736:DNA Adducts; D004151:Dipeptides; C530805:GWL 78; D001569:Benzodiazepines