PMID:22581770

Citation	Culyba, M, Hwang, Y, Attar, S, Madrid, PB, Bupp, J, Huryn, D, Sanchez, L, Grobler, J, Miller, MD and Bushman, FD (2012) Bulged DNA substrates for identifying poxvirus resolvase inhibitors. Nucleic Acids Res. 40:e124
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.
Links	PubMed PMC3439875 Online version:10.1093/nar/gks325
Keywords	DNA/chemistry; DNA/metabolism; DNA Cleavage; DNA, Cruciform/metabolism; Enzyme Assays/methods; Enzyme Inhibitors/chemistry; Enzyme Inhibitors/pharmacology; Fluorescence Polarization; Fowlpox virus/enzymology; Holliday Junction Resolvases/metabolism; Naphthyridines/chemistry; Naphthyridines/pharmacology; Small Molecule Libraries; Structure-Activity Relationship
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
FOWPN:RUVV		GO:0008821: crossover junction endodeoxyribonuclease activity	ECO:0000314:		F		Fig 1	complete CACAO 6834
FOWPN:RUVV	enables	GO:0008821: crossover junction endodeoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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