INTERACTION OF CALICHEAMICIN WITH DUPLEX DNA - ROLE OF THE OLIGOSACCHARIDE DOMAIN AND IDENTIFICATION OF MULTIPLE BINDING MODES

A 1:1 complex between calicheamicin theta(1)(I) (1) (an S-acetyl derivative of calicheamicin gamma(1)(I), 2) and the duplex d(GCATCCTAGC).d(GCTAGGATGC) has been studied by 1D and 2D H-1 NMR spectroscopy to establish the molecular basis for binding and the sequence selectivity of the interaction between this potent antitumor antibiotic and DNA. Complete sequence-specific assignments have been obtained for all drug and DNA protons (except 5'H and 5''H DNA protons). A complex between the same duplex and the calicheamicin gamma(1)(I), oligosaccharide moiety (3) has also been examined in detail by H-1 NMR spectroscopy, and the results are virtually identical. Comparative analysis of the complex formed with and without aglycone demonstrates the critical role of the oligosaccharide domain in the molecular interactions leading to binding and sequence-specific recognition in the minor groove of the DNA duplex. A structural model for the complex was generated by refining manually docked initial structures using molecular dynamics calculations with NMR-derived distance restraints. A total of 194 DNA-DNA distance constraints (including 16 H-bond constraints), 13 drug-drug intra- and interresidue distance constraints, and 15 drug-DNA distance constraints were included in the calculations. Structures refined starting from standard A-form and B-form DNA geometries converged to a root mean square deviation of 1.12 Angstrom for the well-defined regions of the complex. The current structural model is stabilized by several specific interactions including H-bonds and favorable van der Waals interactions. Temperature-dependence studies of the H-1 NMR spectrum revealed that calicheamicin theta(1)(I) (1) binds in two different modes within the d(TCCT).d(AGGA) recognition site; two unequally populated species in slow exchange on the NMR time scale are observed at low temperature. The lifetimes of the major and minor binding modes were determined to be <19 and <6 ms, respectively, at 320 K, with an activation energy (Delta G(double dagger)) of ca. 15 kcal/mol. We propose that this phenomenon is due to a movement of the aglycone portion of the drug in the DNA minor groove. This dynamic process thus provides a critical new insight to explain the observed DNA cleavage properties of calicheamicin.