title: High-throughput multiplex flow cytometry screening for botulinum neurotoxin type a light chain protease inhibitors.
abstract: Given their medical importance, proteases have been studied by diverse approaches and screened for small molecule protease inhibitors. 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.
authors.affiliation: The Center for Molecular Discovery and Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131, USA. msaunders@salud.unm.edu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Saunders
authors.forename: Steven W
authors.initials: SW
authors.lastname: Graves
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
mesh_terms: D000942:Antigens, Bacterial; D001393:Azoles / Q000494:pharmacology; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D054833:Isoindoles; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D008863:Microspheres; D016566:Organoselenium Compounds / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*
chemical_list: D000942:Antigens, Bacterial; D001393:Azoles; D001427:Bacterial Toxins; D054833:Isoindoles; D016566:Organoselenium Compounds; D011480:Protease Inhibitors; C030325:anthrax toxin; C042986:ebselen; D045726:Metalloproteases; D019274:Botulinum Toxins, Type A