Human topoisomerase I inhibition: Docking camptothecin and derivatives into a structure-based active site model

Human topoisomerase I (top 1) is an important target for anti-cancer drugs, which include camptothecin (CPT) and its derivatives. To elucidate top 1 inhibition in vitro, we made a series of duplex DNA substrates containing a deoxyadenosine stereospecifically modified by a covalent adduct of benzo[a]pyrene (BaP) diol epoxide [Pommier, Y., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10739-10744]. The known orientation of the hydrocarbon adduct in the DNA duplex relative to the top 1 cleavage site, in combination with a top 1/DNA crystal structure [Redinbo, M. R., et al. (1998) Science 279, 1504-1513], was used to construct a structure-based model to explain the in vitro top 1 inhibition results obtained with adducted DNA duplexes. Here we experimentally determined that the lactone form of CPT was stabilized by an irreversible top 1/DNA covalent complex. We removed the BaP moiety from the DNA in the published model, and docked the lactone forms of CPT and derivatives into the top 1/DNA active site cavity. The docked ligands were minimized, and interaction energy scores between the ligands and the top 1/DNA complex were determined. CPT docks perpendicular to the DNA backbone, projects outward 7 from the major groove, and makes a network of potential H-bonds with the active site DNA and top 1 residues, including Arg364, Lys532, and Asn722. The results are consistent with the known structureactivity relationships of CPT and derivatives. In addition, the model proposed a novel top 1/N352A "resistance" mutation for 10-OH derivatives of CPT. The in vitro biochemical characterization of the top 1/N352A mutant supported the model.