{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T09:57:41Z","timestamp":1781690261078,"version":"3.54.5"},"reference-count":88,"publisher":"Oxford University Press (OUP)","issue":"11","license":[{"start":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T00:00:00Z","timestamp":1781654400000},"content-version":"vor","delay-in-days":9,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["1R21AI125973"],"award-info":[{"award-number":["1R21AI125973"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["1R21AI178134"],"award-info":[{"award-number":["1R21AI178134"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006108","name":"National Center for Advancing Translational Sciences","doi-asserted-by":"publisher","award":["1R41TR005250"],"award-info":[{"award-number":["1R41TR005250"]}],"id":[{"id":"10.13039\/100006108","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,6,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Antimicrobial resistance is a major global public health threat, yet no new antibiotic class has been introduced in over 30 years. Fluoroquinolones (FQs), a type of bacterial DNA gyrase poison, are potent antibiotics which stabilizes gyrase\u2013DNA cleavage complexes and therefore cause lethal DNA double-stranded breaks. However, rising FQ resistance combined with adverse effects highlight the need to identify new chemical scaffolds for gyrase poisons. Discovery efforts in this area have been limited by the lack of robust high-throughput screening (HTS) assays. Here, we describe a fluorescence-based, T5 exonuclease (T5E)-assisted DNA cleavage assay that exploits the formation of multiple gyrase\u2013DNA cleavage complexes on a single plasmid. When stabilized by a gyrase poison, these complexes are denatured with sarkosyl and processed by proteinase K to generate small linear DNA fragments, which are selectively degraded by T5E, thereby decreasing fluorescence upon SYBR Green staining. This assay was miniaturized into an automated 1536-well ultra-HTS format and used to screen 7415 compounds, identifying numerous potent gyrase poisons, predominantly FQs. We also discovered a new gyrase poison, Pyr-AMC. Biochemical assays and molecular dynamic simulation demonstrate that a single Pyr-AMC molecule binds the hydrophobic gepotidacin-binding pocket of gyrase, stabilizing cleavage complexes, and trapping gyrase-mediated DNA cleavages.<\/jats:p>","DOI":"10.1093\/nar\/gkag610","type":"journal-article","created":{"date-parts":[[2026,6,7]],"date-time":"2026-06-07T11:32:53Z","timestamp":1780831973000},"source":"Crossref","is-referenced-by-count":0,"title":["A high-throughput fluorescent DNA cleavage assay for discovering bacterial DNA gyrase poisons"],"prefix":"10.1093","volume":"54","author":[{"given":"Matthew","family":"Dias","sequence":"first","affiliation":[{"name":"Biomolecular Sciences Institute, Florida International University , Miami, FL 33199 ,","place":["United States"]},{"name":"Department of Chemistry and Biochemistry, Florida International University , Miami, FL 33199 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thirunavukkarasu","family":"Annamalai","sequence":"additional","affiliation":[{"name":"Biomolecular Sciences Institute, Florida International University , Miami, FL 33199 ,","place":["United States"]},{"name":"Department of Chemistry and Biochemistry, Florida International University , Miami, FL 33199 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuka","family":"Otsuka","sequence":"additional","affiliation":[{"name":"The Wertheim UF Scripps HTS Center, Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology , Jupiter, FL 33458 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophia","family":"Leng","sequence":"additional","affiliation":[{"name":"Biomolecular Sciences Institute, Florida International University , Miami, FL 33199 ,","place":["United 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,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin","family":"Shumate","sequence":"additional","affiliation":[{"name":"The Wertheim UF Scripps HTS Center, Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology , Jupiter, FL 33458 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Louis","family":"Scampavia","sequence":"additional","affiliation":[{"name":"The Wertheim UF Scripps HTS Center, Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology , Jupiter, FL 33458 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy P","family":"Spicer","sequence":"additional","affiliation":[{"name":"The Wertheim UF Scripps HTS Center, Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology , Jupiter, FL 33458 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9024-1216","authenticated-orcid":false,"given":"Fenfei","family":"Leng","sequence":"additional","affiliation":[{"name":"Biomolecular Sciences Institute, Florida International University , Miami, FL 33199 ,","place":["United States"]},{"name":"Department of Chemistry and Biochemistry, Florida International University , Miami, FL 33199 ,","place":["United States"]},{"name":"Top Biosciences, LLC, 7405 SW 157th Terrace , Palmetto Bay, FL 33157 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,6,17]]},"reference":[{"key":"2026061705270596000_B1","doi-asserted-by":"publisher","first-page":"635","DOI":"10.1146\/annurev.bi.65.070196.003223","article-title":"DNA topoisomerases","volume":"65","author":"Wang","year":"1996","journal-title":"Annu Rev 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