Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health
May 2nd, 2016 – Spring Seminar
Time and location: Noon at Public Policy 105
“Single-molecule measurements of human topoisomerase IB and its inhibition by chemotherapeutics“
Host: Dr. Minjoung Kyoung
Human type IB topoisomerases relax excessive DNA supercoiling associated with replication and transcription. Human nuclear type IB topoisomerase (nTop1) inhibitors are widely used and powerful anticancer agents. We have developed and validated 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 (indenoisoquinoline derivatives currently in phase I trials), 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 demonstrated a strong correlation between the binding time of 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.
Seol, Yeonee, et al. “Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors.” Molecular cancer therapeutics 14.11 (2015): 2552-2559.
Seol, Yeonee, et al. “A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity.” Proceedings of the National Academy of Sciences 109.40 (2012): 16125-16130.