Identification of Small Molecules against Botulinum Neurotoxin B Binding to Neuronal Cells at Ganglioside GT1b Binding Site with Low to Moderate Affinity.

摘要

Clostridium botulinum bacteria produce botulinum neurotoxin (BoNT) which can be developed as a bioweapon. Currently no FDA-licensed prophylactic product is available in the USA against botulism. Small molecules are being developed for use as inhibitors against the proteolytic activity of the toxin, but we are investigating an alternative strategy where small molecule inhibitors may prevent intoxication by disrupting the binding process of the toxin to neuronal cells. Recently, four potential binding sites or pockets on BoNT serotype B (BoNT/B) for the trisaccharide GT1b were identified from the x-ray crystal structure of the BoNT/B/trisaccharide (GT1b) complex (PDB id: 1F31). To develop inhibitors for these sites we designed a five-point 3D stereo-electronic pharmacophore model for Pockets 1 and 2. Queries were made of compound libraries from the NCI/DTP (National Cancer Institute/Developmental Therapeutics Program) based on features of the pharmacophore model. Library compounds were screened in silico to identify those that could dock into Pocket 1 or a combination of Pockets 1 and 2. We rank-ordered the compounds based on consensus scoring. Seventy compounds were identified by the model. Available compounds were tested in a cell-based ELISA binding inhibition assay. With this assay we identified two lead compounds that prevented the binding of BoNT/B binding domain (BD) to GT1b. These initial compounds demonstrate the utility of this approach and could form the basis of further discovery and refinement for the design of more potent small molecule inhibitors