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Human topoisomerase I inhibition: docking camptothecin and derivatives into a structure-based active site model
Gary S Laco1, Jack R Collins, Brian T Luke
1Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Human topoisomerase I (top1) is an important target for anti-cancer drugs, which include camptothecin (CPT) and its derivatives. To elucidate top1 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 top1 cleavage site, 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 obtained with adducted DNA duplexes. Here we experimentally determined that the lactone form of CPT was stabilized by an irreversible top1/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 top1/DNA active site cavity. The docked ligands were minimized, and interaction energy scores between the ligands and the top1/DNA complex were determined. CPT docks perpendicular to the DNA backbone, projects outward from the major groove, and makes a network of potential H-bonds with the active site DNA and top1 residues, 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 the top1/N352A mutant supported the model.
Insights
Camptothecin (CPT) stabilizes human topoisomerase I (top1) covalent complexes. This study models CPT binding to top1-DNA, revealing interactions and predicting resistance mutations for anti-cancer drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Medicinal Chemistry
Background:
- Human topoisomerase I (top1) is a validated anti-cancer drug target.
- Camptothecin (CPT) and its derivatives are key top1-inhibiting anti-cancer agents.
- Understanding drug-target interactions is crucial for developing effective cancer therapies.
Purpose of the Study:
- To elucidate the in vitro inhibition mechanism of topoisomerase I (top1) by camptothecin (CPT).
- To develop a structure-based model of CPT and its derivatives interacting with the top1/DNA complex.
- To investigate potential resistance mechanisms to CPT derivatives.
Main Methods:
- Construction of DNA substrates with benzo[a]pyrene (BaP) adducts.
- Structure-based modeling of CPT and derivatives docked into the top1/DNA active site.
- In vitro biochemical characterization of top1 mutants.
Main Results:
- CPT stabilizes an irreversible top1/DNA covalent complex.
- CPT docks perpendicular to the DNA, interacting with top1 residues (Arg364, Lys532, Asn722) and DNA.
- A novel top1/N352A resistance mutation model for 10-OH CPT derivatives was proposed and supported by experimental data.
Conclusions:
- The study provides a detailed structural model for CPT inhibition of top1.
- The findings explain structure-activity relationships of CPT derivatives.
- The identified resistance mutation offers insights for future drug design.