{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T16:13:25Z","timestamp":1791562405461,"version":"4.3.4"},"reference-count":44,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T00:00:00Z","timestamp":1696982400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Drug Development Research"],"published-print":{"date-parts":[[2024,2]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The use of cell growth\u2010based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell\u2010based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case <jats:sc>l<\/jats:sc>\u2010methionine <jats:italic>S<\/jats:italic>\u2010adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing <jats:italic>Escherichia coli<\/jats:italic> strain that carries a deletion of <jats:italic>metK<\/jats:italic>, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied <jats:italic>S<\/jats:italic>\u2010adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species\u2010selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog\u2010selective inhibitors of this enzyme family, some of which target the protozoal human pathogen <jats:italic>Cryptosporidium parvum<\/jats:italic>.<\/jats:p>","DOI":"10.1002\/ddr.22122","type":"journal-article","created":{"date-parts":[[2023,10,12]],"date-time":"2023-10-12T01:33:14Z","timestamp":1697074394000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A growth\u2010based assay using fluorescent protein emission to screen for <i>S<\/i>\u2010adenosylmethionine synthetase inhibitors"],"prefix":"10.1002","volume":"85","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3117-9168","authenticated-orcid":false,"given":"Ronald E.","family":"Viola","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry University of Toledo Toledo Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gwenn G.","family":"Parungao","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of Toledo Toledo Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0873-0724","authenticated-orcid":false,"given":"Robert M.","family":"Blumenthal","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, and Program in Bioinformatics University of Toledo Health Sciences Campus Toledo Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,10,11]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12033-010-9251-z"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12015-016-9670-8"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4509"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2020.07.024"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.44.070175.002251"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.8303295"},{"key":"e_1_2_10_8_1","volume-title":"S\u2010adenosylmethionine\u2010dependent methyltransferases: Structures and functions","author":"Cheng X.","year":"2000"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/2211-5463.13312"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.295857"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1359-6446(03)02831-9"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2021.689401"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.187.16.5719-5722.2005"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1998.2677"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2004.03.007"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216-010-3651-6"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1177\/1087057110381383"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bb.24.060195.002201"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-7799(97)01166-9"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0075991"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens8030116"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2016.07.005"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.3390\/s21030795"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201609615"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-008-8516-1"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200900777"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmc.2016.08.003"},{"key":"e_1_2_10_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0163-7258(96)00197-0"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.enzmictec.2018.06.016"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216-010-3933-z"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0064753"},{"key":"e_1_2_10_32_1","first-page":"290","article-title":"Structure of a critical metabolic enzyme: S\u2010adenosylmethionine synthetase from Cryptosporidium parvum","volume":"5","author":"Ohren J.","year":"2019","journal-title":"Acta Crystallographica. SF7"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.000565"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.260370504"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2003.11.022"},{"key":"e_1_2_10_36_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00982-21"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1002\/bies.10237"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13073-020-00782-x"},{"key":"e_1_2_10_39_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)41382-3"},{"key":"e_1_2_10_40_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.185.10.3031-3035.2003"},{"key":"e_1_2_10_41_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M207727200"},{"key":"e_1_2_10_42_1","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.000034"},{"key":"e_1_2_10_43_1","doi-asserted-by":"publisher","DOI":"10.1039\/D2CS00419D"},{"key":"e_1_2_10_44_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.REV120.009402"},{"key":"e_1_2_10_45_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.abb.2013.05.008"}],"container-title":["Drug Development Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/ddr.22122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T06:33:03Z","timestamp":1760423583000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/ddr.22122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,11]]},"references-count":44,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2024,2]]}},"alternative-id":["10.1002\/ddr.22122"],"URL":"https:\/\/doi.org\/10.1002\/ddr.22122","archive":["Portico"],"relation":{},"ISSN":["0272-4391","1098-2299"],"issn-type":[{"value":"0272-4391","type":"print"},{"value":"1098-2299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,11]]},"assertion":[{"value":"2023-06-14","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-10-02","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-10-11","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e22122"}}