Bulged DNA substrates for identifying poxvirus resolvase inhibitors |
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Authors: | Culyba Matthew Hwang Young Attar Sana Madrid Peter B Bupp James Huryn Donna Sanchez Luis Grobler Jay Miller Michael D Bushman Frederic D |
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Institution: | Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, Center for Infectious Disease and Biodefense Research, Bioscience Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323 and Department of Infectious Diseases, Merck Research Laboratories, West Point, PA 19486, USA. |
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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. |
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