Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors.
Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anticancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors.
Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, Maryland.
Yeonee
Y
Seol
Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.
Hongliang
H
Zhang
Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.
Keli
K
Agama
Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.
Nicholas
N
Lorence
Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. neumankc@mail.nih.gov pommier@nih.gov.
Yves
Y
Pommier
Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, Maryland. neumankc@mail.nih.gov pommier@nih.gov.
Keir C
KC
Neuman
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D052117:Benzodioxoles / Q000737:chemistry / Q000494:pharmacology; D002166:Camptothecin / Q000737:chemistry / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D053837:DNA Cleavage / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D013379:Substrate Specificity; D059004:Topoisomerase I Inhibitors / Q000737:chemistry / Q000494:pharmacology*
D000970:Antineoplastic Agents; D052117:Benzodioxoles; D003374:Coumarins; D004278:DNA, Superhelical; D006576:Heterocyclic Compounds, 4 or More Rings; D007546:Isoquinolines; C553898:NSC 724998; D059004:Topoisomerase I Inhibitors; C455554:lamellarin D; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; C578795:indimitecan; D002166:Camptothecin