Use of a recombinant fluorescent substrate with cleavage sites for all botulinum neurotoxins in high-throughput screening of natural product extracts for inhibitors of serotypes A, B, and E

Abstract

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

FIG. 1.

A schematic representation of the recombinant fluorescent BoNT substrate GFPSV (not to scale) covalently bound through an N-ethylsuccinimide linkage to a plate well. Cleavage sites for the various BoNT serotypes are indicated by arrows.

FIG. 2.

Hydrolysis of the immobilized recombinant substrate by BoNT protease activities. Each point is the average from triplicate determinations, and in all cases, standard deviations were less than ±10%. (A) Blank (buffer only), A-Lc, and B-Lc. (B) BoNT D and F holotoxins and E-Lc.

FIG. 3.

Representative high-throughput assays of BoNT A protease activity in the presence of extracts from plate 96110120. (A) Selected wells from row B. (B) Selected wells from row G.

FIG. 4.

Fractionation of Terminalia brownii root bark extract by reverse-phase HPLC. (A) Chromatogram of the column effluent monitored at 210 nm. Peaks 1 and 2 inhibited BoNT A protease activity. (B) Absorbance spectra of peaks 1 and 2. (C) Absorbance spectrum of myricetin.