A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators |
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Authors: | Dejan Cagli? Kristin M Bompiani Michelle C Krutein Petr ?apek Tobin J Dickerson |
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Institution: | 1.Department of Chemistry, The Scripps Research Institute;2.Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute |
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Abstract: | Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds. |
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Keywords: | Chemistry Issue 82 BoNT/A botulinum neurotoxin high-throughput screening FRET inhibitor FRET peptide substrate activator |
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