title: A high content imaging assay for identification of Botulinum neurotoxin inhibitors.
abstract: Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.
authors.affiliation: Perkin Elmer Inc.; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases; krishna.p.kota.ctr@mail.mil.
authors.forename: Krishna P
authors.initials: KP
authors.lastname: Kota
authors.affiliation: Henry M. Jackson Foundation; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases; DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI), Telemedicine and Advanced Technology Research Center (TATRC), US Army Medical Research and Materiel Command (USAMRMC).
authors.forename: Veronica
authors.initials: V
authors.lastname: Soloveva
authors.affiliation: The Geneva Foundation; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Wanner
authors.affiliation: ORISE; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases.
authors.forename: Glenn
authors.initials: G
authors.lastname: Gomba
authors.affiliation: The Geneva Foundation; Frederick National Laboratory for Cancer Research; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases.
authors.forename: Erkan
authors.initials: E
authors.lastname: Kiris
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases.
authors.forename: Rekha G
authors.initials: RG
authors.lastname: Panchal
authors.affiliation: Henry M. Jackson Foundation; Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases; DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI), Telemedicine and Advanced Technology Research Center (TATRC), US Army Medical Research and Materiel Command (USAMRMC).
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Kane
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases.
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000379:methods; D051379:Mice; D009046:Motor Neurons / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D050825:Synaptosomal-Associated Protein 25 / Q000032:analysis* / Q000378:metabolism
chemical_list: C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A