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A Solid Phase Assay for Topoisomerase I interfacial Poisons and Catalytic Inhibitors

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Date Issued:
2011
Abstract/Description:
We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a 'bimodal' readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.
Title: A Solid Phase Assay for Topoisomerase I interfacial Poisons and Catalytic Inhibitors.
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Name(s): Cyril Sagayaraj, Vidusha, Author
Muller, Mark, Committee Chair
Zhao, Jihe, Committee Member
Chakrabarti, Debopam, Committee Member
, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2011
Publisher: University of Central Florida
Language(s): English
Abstract/Description: We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a 'bimodal' readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.
Identifier: CFE0004473 (IID), ucf:49304 (fedora)
Note(s): 2011-12-01
M.S.
Medicine, Molecular Biology and Microbiology
Masters
This record was generated from author submitted information.
Subject(s): Eukaryotic topoisomerase I -- High-Throughput screening -- Solid Phase Assay -- Anti cancer compounds -- DNA binding proteins
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0004473
Restrictions on Access: campus 2013-06-15
Host Institution: UCF

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