FIG 1.

Substrate design for the HTS endopeptidase assays. (A) Schematic of the substrate SNAP-25 (aa 1 to 206) and proteolytic fragments generated following cleavage of SNAP-25 at residues Q197 and R198 by BoNT/A. The residues shown in red were used for FRET peptide substrate development. (B) Sequence of FRET peptide substrate used for HTS of inhibitors. (C) Full-length substrate (GST [26.31 kDa, purple], SNAP25a [23.82 kDa, blue], GFP [26.94 kDa, green], SNAG [77.03 kDa]) for confirmation of inhibition by NPP. SNAG cleaves into 48.73-kDa and 28.33-kDa fragments when incubated with BoNT/A and into 46.78-kDa and 30.27-kDa fragments when incubated with BoNT/E. EGCP, enhanced green fluorescent protein.