Rapid microtiter assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation.

Abstract

We have developed microtiter assays for detecting catalysis by type IB topoisomerases and retroviral integrases. Each assay employs model DNA substrates containing biotin in one strand and digoxigenin in another. In each case action of the enzyme results in the formation of a single DNA strand containing both groups. This allows the reaction product to be quantified by capturing biotinylated product DNA on avidin-coated plates followed by detection using an anti-digoxigenin ELISA. The order of addition of reactants and inhibitors can be varied to distinguish effects of test compounds on different steps in the reaction. These assays were used to screen compound libraries for inhibitors active against mammalian topoisomerase or HIV integrase. We identified (-)-epigallocatechin 3-O:-gallate, as a potent inhibitor of religation by mammalian topoisomerase (IC(50) of 26 nM), potentially explaining the anti-cancer properties previously attributed to this compound. New integrase inhibitors were also identified. A similar strategy may be used to develop microtiter assays for many further DNA modifying enzymes.

• Authors: Y Hwang, D Rhodes, F Bushman
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 28 Issue 24 Pg. 4884-92 (Dec 15 2000) ISSN: 1362-4962 [Electronic] England
• PMID: 11121479 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antineoplastic Agents
• Enzyme Inhibitors
• HIV Integrase Inhibitors
• Topoisomerase I Inhibitors
• Avidin
• Catechin
• epigallocatechin gallate
• HIV Integrase
• DNA Topoisomerases, Type I

Topics

• Animals
• Antineoplastic Agents (isolation & purification, pharmacology)
• Avidin (metabolism)
• Base Sequence
• Biotinylation
• Catalysis
• Catechin (analogs & derivatives, isolation & purification, pharmacology)
• DNA Topoisomerases, Type I (classification, metabolism)
• Drug Evaluation, Preclinical (methods)
• Enzyme Inhibitors (isolation & purification, pharmacology)
• Enzyme-Linked Immunosorbent Assay (methods)
• HIV Integrase (metabolism)
• HIV Integrase Inhibitors (isolation & purification, pharmacology)
• HIV-1 (drug effects, enzymology)
• Inhibitory Concentration 50
• Molluscum contagiosum virus (drug effects, enzymology)
• Reproducibility of Results
• Time Factors
• Topoisomerase I Inhibitors