title: Bulged DNA substrates for identifying poxvirus resolvase inhibitors.
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.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Culyba
authors.forename: Young
authors.initials: Y
authors.lastname: Hwang
authors.forename: Sana
authors.initials: S
authors.lastname: Attar
authors.forename: Peter B
authors.initials: PB
authors.lastname: Madrid
authors.forename: James
authors.initials: J
authors.lastname: Bupp
authors.forename: Donna
authors.initials: D
authors.lastname: Huryn
authors.forename: Luis
authors.initials: L
authors.lastname: Sanchez
authors.forename: Jay
authors.initials: J
authors.lastname: Grobler
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D045566:DNA, Cruciform / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005454:Fluorescence Polarization; D005587:Fowlpox virus / Q000201:enzymology*; D045565:Holliday Junction Resolvases / Q000378:metabolism*; D009287:Naphthyridines / Q000737:chemistry / Q000494:pharmacology; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship
chemical_list: C556786:1-hydroxy-1,8-naphthyridin-2(1H)-one; D045566:DNA, Cruciform; D004791:Enzyme Inhibitors; D009287:Naphthyridines; D054852:Small Molecule Libraries; D004247:DNA; D045565:Holliday Junction Resolvases