Nuclear Magnetic Resonance Solution Structures of Inter- and Intrastrand Adducts of DNA Cross-Linker SJG-136

Abstract

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials for the treatment of malignant disease. Previous studies on the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, typified by SJG-136 and DSB-120 (2), have shown that these planar ligands react with the exocyclic NH(2) groups of two guanine bases in the base of the minor groove of DNA to form an irreversible interstrand cross-linked sequence-specific adduct. Using high-field NMR, we have characterized and modeled the previously predicted interstrand duplex adduct formed by SJG-136 with the self-complementary 5'-d(CICGATCICG)(2) duplex (4). This first SJG-136 NMR-refined adduct structure has been compared with previous high-field NMR studies of the adducts of the closely related PBD dimer DSB-120 with the same duplex and of the adduct of tomaymycin (3) formed with 5'-d(ATGCAT)(2). Surprisingly, the SJG-136 duplex adduct appears to be more closely related to the tomaymycin adduct than to the DSB-120 adduct with respect of the orientation and depth of insertion of the ligand within the minor groove. The intrastrand duplex adduct formed in the reaction of SJG-136 with the noncomplementary 5'-d(CTCATCAC)·(GTGATGAG) duplex (5) has also been synthesized and modeled. In this duplex adduct, the nature of the cross-link was confirmed, the central guanines were identified as the sites of alkylation, and the stereochemical configuration at C11 at both ends of the SJG-136 molecule was determined to be S. The NMR-refined solution structures produced for the intrastrand adduct confirm the previously proposed structure (which was based solely on mass spectroscopy). Both the inter- and intrastrand SJG-136 duplex adducts form with minimal distortion of the DNA duplex. These observations have an impact on the proposal for the mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and, potentially, on the type of pharmacodynamic assay to be used in clinical trials. SGJ-136 is currently in phase II clinical trials with several groups working on both dimeric cross-linking agents and monoalkylating ligands based on the PBD alkylating moiety. This study suggests subtle differences between the DNA binding of SJG-136 and the C2 unsubstituted analogue DSB-120 that are likely to be the origins of the differences in potency. Confirmation of the stereochemical configuration at the C11 position (particularly in the intrastrand adduct) provides confirmation of binding orientation that was previously only speculation in the HPLC MS study. Together, these observations are likely to be of value in the development of third-generation PBD-based cross-linkers and monoalkylating analogues.