Reductive therapeutic strategy to prevent BONT intoxication

Abstract

The light chain (LC) of BoNT is brought into the cell in association with the heavy chain (HC) through the HC/receptor endocytotic mechanism. The LC-HC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits yield LC endopeptidase. This requirement suggests a strategy of reductive prophylaxis to disrupt the disulfide bond prior to target cell binding. We examined the utility of tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), a relatively non-toxic, non-sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercaptan reducing agents. TCEP was as effective as DTT for activation of BoNT/B in vitro. TCEP was then tested as a pretreatment to prevent BoNT intoxication in SHSY-5Y human neuroblastoma cells. Vesicle trafficking was measured following K+ stimulation of cells preloaded with [3 H]-noradrenaline. Preincubation of BoNT with 1 mM TCEP maximally protected against inhibition release, achieving 72% of untreated controls, and was not toxic at this level. The protective effect was likely due to the sparing of SNARE proteins as VAMP-2 levels were protected. Since disulfide bond coupling between toxin subunits is a general motif for many toxins that require reduction to separate subunits, our results suggest that TCEP could be a promising means to protect against these toxins by preventing cell penetration. Support, DTRA#3.10017_06_WR_B.