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Here, we present a multiplexed microsphere-based protease assay that uses high-throughput flow cytometry to screen for inhibitors of the light chain protease of botulinum neurotoxin type A (BoNTALC). Our assay uses a full-length substrate and several deletion mutants screened in parallel to identify small molecule inhibitors. The use of multiplex flow cytometry has the advantage of using full-length substrates, which contain already identified distal-binding elements for the BoNTALC, and could lead to a new class of BoNTALC inhibitors. In this study, we have screened 880 off patent drugs and bioavailable compounds to identify ebselen as an in vitro inhibitor of BoNTALC. This discovery demonstrates the validity of our microsphere-based approach and illustrates its potential for high-throughput screening for inhibitors of proteases in general.<\/jats:p>","DOI":"10.1089\/adt.2009.0219","type":"journal-article","created":{"date-parts":[[2009,12,28]],"date-time":"2009-12-28T07:39:47Z","timestamp":1261985987000},"page":"37-46","source":"Crossref","is-referenced-by-count":20,"title":["High-Throughput Multiplex Flow Cytometry Screening for Botulinum Neurotoxin Type A Light Chain Protease Inhibitors"],"prefix":"10.1177","volume":"8","author":[{"given":"Matthew J.","family":"Saunders","sequence":"first","affiliation":[{"name":"The Center for Molecular Discovery and Department of Pathology, University of New Mexico, Albuquerque, New Mexico."},{"name":"The Center for Biomedical Engineering, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico."},{"name":"The National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven W.","family":"Graves","sequence":"additional","affiliation":[{"name":"The Center for Biomedical Engineering, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico."},{"name":"The National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Larry A.","family":"Sklar","sequence":"additional","affiliation":[{"name":"The Center for Molecular Discovery and Department of Pathology, University of New Mexico, Albuquerque, New Mexico."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tudor I.","family":"Oprea","sequence":"additional","affiliation":[{"name":"The Center for Molecular Discovery and Department of Pathology, University of New Mexico, Albuquerque, New Mexico."},{"name":"Division of Biocomputing, Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, New Mexico."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce S.","family":"Edwards","sequence":"additional","affiliation":[{"name":"The Center for Molecular Discovery and Department of Pathology, University of New Mexico, Albuquerque, New Mexico."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"179","published-online":{"date-parts":[[2010,2,1]]},"reference":[{"key":"e_1_3_5_2_2","first-page":"1","volume-title":"Essays in Biochemistry: Proteases in Biology and Medicine","author":"Hooper NM","year":"2002","unstructured":"Hooper NM: Proteases: a primer. 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