Bulged DNA substrates for identifying poxvirus resolvase inhibitors.

Nucleic Acids Research
Matthew J CulybaFrederic D Bushman

Abstract

Resolvase enzymes that cleave DNA four-way (Holliday) junctions are required for poxvirus replication, but clinically useful inhibitors have not been developed. Here, we report an assay for resolvase cleavage activity based on fluorescence polarization (FP) for high-throughput screening and mechanistic studies. Initial analysis showed that cleavage of a fluorescently labeled Holliday junction substrate did not yield an appreciable change in FP, probably because the cleavage product did not have sufficiently increased mobility to yield a strong FP signal. Iterative optimization yielded a substrate with an off-center DNA bulge, which after cleavage released a labeled short stand and yielded a greatly reduced FP signal. Using this assay, 133,000 compounds were screened, identifying 1-hydroxy-1,8-naphthyridin-2(1H)-one compounds as inhibitors. Structure-activity studies revealed functional parallels to Food and Drug Administration (FDA)-approved drugs targeting the related human immunodeficiency virus integrase enzyme. Some 1-hydroxy-1,8-naphthyridin-2(1H)-one compounds showed anti-poxvirus activity.

References

Feb 15, 1995·Structure·W Yang, T A Steitz
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Jul 13, 2000·Proceedings of the National Academy of Sciences of the United States of America·A D GarciaB Moss
Mar 16, 2002·Nature Structural Biology·Scott LovellIvan Rayment
May 9, 2002·Proceedings of the National Academy of Sciences of the United States of America·Jay A GroblerDaria J Hazuda
Jun 20, 2006·Virology·Matthew J CulybaFrederic D Bushman
Dec 13, 2006·Nucleic Acids Research·Michael D HamiltonDavid H Evans
Sep 25, 2007·The Journal of Biological Chemistry·Matthew J CulybaFrederic D Bushman
Nov 14, 2008·The Journal of Biological Chemistry·Matthew J CulybaFrederic D Bushman
Feb 2, 2010·Nature·Stephen HarePeter Cherepanov
Apr 13, 2010·Journal of Molecular Biology·Matthew J CulybaFrederic D Bushman
Sep 28, 2010·Bioorganic & Medicinal Chemistry Letters·Peter D WilliamsJay A Grobler

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Citations

Mar 26, 2016·The Journal of Biological Chemistry·Huiguang LiGregory D Van Duyne
May 29, 2015·Biochemistry·Matthew J CulybaRahul M Kohli

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Methods Mentioned

BETA
electrophoresis
Assay

Software Mentioned

Prism

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