Human Topoisomerase I Inhibition: Docking Camptothecin and Derivatives into a Structure-Based Active Site Model

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• 作者：Gary S. Laco ; Jack R. Collins ; Brian T. Luke ; Heiko Kroth ; Jane M. Sayer ; Donald M. Jerina ; and Yves Pommier
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 5, 2002
• 年：2002
• 卷：41
• 期：5
• 页码：1428 - 1435
• 全文大小：189K
• 年卷期：v.41,no.5(February 5, 2002)
• ISSN：1520-4995

文摘

Human topoisomerase I (top1) is an important target for anti-cancer drugs, which includecamptothecin (CPT) and its derivatives. To elucidate top1 inhibition in vitro, we made a series of duplexDNA 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 top1 cleavagesite, in combination with a top1/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 top1 inhibition results obtainedwith adducted DNA duplexes. Here we experimentally determined that the lactone form of CPT wasstabilized by an irreversible top1/DNA covalent complex. We removed the BaP moiety from the DNA inthe published model, and docked the lactone forms of CPT and derivatives into the top1/DNA active sitecavity. The docked ligands were minimized, and interaction energy scores between the ligands and thetop1/DNA complex were determined. CPT docks perpendicular to the DNA backbone, projects outwardfrom the major groove, and makes a network of potential H-bonds with the active site DNA and top1residues, including Arg364, Lys532, and Asn722. The results are consistent with the known structure-activity relationships of CPT and derivatives. In addition, the model proposed a novel top1/N352A"resistance" mutation for 10-OH derivatives of CPT. The in vitro biochemical characterization of thetop1/N352A mutant supported the model.