Sequence-Selective Recognition of Duplex DNA through Covalent Interstrand Cross-Linking: Kinetic and Molecular Modeling Studies with Pyrrolobenzodiazepine Dimers

摘要

Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH_2)_n-O-C8' diether linkages (n=3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n=3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n=5) can span an extra base pair and cross-link the 5'-Pu-Ga(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n=3 (2a) > n=5 (2c) > n=6 (2d) > n=4(2b). The n=3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n=3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h~(-1) muM~(-1) at 37 deg C, respectively), whereas the n=4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h~(-1) muM~(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to cofigurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.